Charge-dynamic polymers and delivery of anionic compounds

ABSTRACT

The present invention provides dynamic charge state cationic polymers that are useful for delivery of anionic molecules. The dynamic charge state cationic polymers are designed to have cationic charge densities that decrease by removal of removable functional groups from the polymers. The present invention also provides interpolyelectrolyte complexes containing the polymers complexed to a polyanion. Methods for using the interpolyelectrolyte complexes to deliver anionic compounds are also provided.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No.60/486,107 filed Jul. 9, 2003, the entire disclosure of which isincorporated herein by reference and for all purposes as if fully setforth herein.

FIELD OF THE INVENTION

The present invention relates to dynamic charge state cationic polymersthat are useful for delivery of anionic molecules and methods using thepolymers.

BACKGROUND OF THE INVENTION

The safe and efficient delivery of DNA to cells presents a formidablechallenge and an obstacle to the clinical success of gene therapy.Anderson, W. F. (1998) Human Gene Therapy. Nature, 392 Suppl. 25-30;Verma, I. M.; Somia, N. (1997) Gene Therapy—Promises, Problems, andProspects. Nature, 389, 239-242; Crystal, R. G. (1995) Transfer of Genesto Humans: Early Lessons and Obstacles to Success. Science, 270,404-410. Synthetic polymers have been investigated widely as genedelivery agents and are generally viewed as long-term alternatives toviruses due to their low immunogenicities and the ease with which theycan be structurally modified. Luo, D.; Saltzman, W. M. (2000) SyntheticDNA Delivery Systems. Nat. Biotechnol., 18, 33-37. Cationic polymers areparticularly useful in this context because they form conjugates withnegatively charged DNA, and the incorporation of new design elementsinto cationic polymers has resulted in advances toward functional genedelivery systems. Despite extensive work, however, polymers remain farless efficient than their viral counterparts.

For efficient gene transfer and expression to occur, a gene deliveryagent (or vector) should overcome numerous intracellular barriers totransfection. Luo, D.; Saltzman, W. M. (2000) Synthetic DNA DeliverySystems. Nat. Biotechnol., 18, 33-37. For example, a vector should beable to: 1) condense DNA into stabilized, nanometer-scale structures, 2)target cells and stimulate internalization, 3) prevent the degradationof DNA inside the cell, 4) target the cell nucleus, and 5) release DNAin the nucleus so that it is available for transcription. Progress hasbeen made toward many of these barriers—cationic polymers are used tocondense DNA into 50 to 200 nm particles (barrier 1), conjugation withcell-specific ligands can be used to target complexes and stimulateuptake (barrier 2), the incorporation of pH-buffering functionality intosynthetic polymers provides protection against acidic intracellularenvironments (barrier 3), and the nuclear membrane has been “breached”(barrier 4). Kabanov, A. V.; Feigner, P. L.; Seymour, L. W., inSelf-Assembling Complexes for Gene Delivery: From Laboratory to ClinicalTrial, John Wiley and Sons, New York, 1998; Putnam, D.; Gentry, C. A.;Pack, D. W.; Langer, R. (2001) Polymer-Based Gene delivery with LowCytotoxicity By a Unique Balance of Side-Chain Termini. Proc. Natl.Acad. Sci. USA, 98, 1200-1205; Midoux, P.; Monsigny, M. (1999) EfficientGene Transfer by Histidylated Polylysine/pDNA Complexes. BioconjugateChem., 10, 406-411; Boussif, O.; Lezoualc'H, F.; Zanta, M. A.; Mergny,M. D.; Scherman, D.; Demeneix, B.; Behr, J. P. (1995) A Versatile vectorfor Gene and Oligonucleotide Transfer Into Cells in Culture and InVivo—Polyethyleneimine Proc. Natl. Acad. Sci. USA, 92, 7297-7301; Benns,J. M.; Choi, J.; Mahato, R. I.; Park, J.; Kim, S. W. (2000) pH-sensitiveCationic Polymer Gene Delivery Vehicle:N-Ac-poly(L-histidine)-graft-poly(L-lysine) Comb Shaped Polymer.Bioconjugate Chem., 11, 67-645; Wolff, J. A.; Sebestyén, M. G. (2001)Nuclear Security Breached. Nature Biotechnol., 19, 1118-1120; Rebuffat,A.; Bernasconi, A.; Ceppi, M.; Wehrli, H.; Verca, S. B.; Ibrahim, M.;Frey, B. M.; Frey, F. J.; Rusconi, S. (2001) Selective Enhancement ofGene transfer by Steroid-Mediated Gene Delivery. Nature Biotechnol., 9,1155-1161. These recent successes have fueled hopes of a “grand design”in which individual design elements could be assembled to createsynthetic vectors that functionally mimic viruses. Wolff, J. A. (2002)The “Grand” Problem of Synthetic Delivery. Nature Biotechnol., 20,768-769. However, the breach of early barriers to transfection simplyplaces increased significance on downstream barriers, and the design ofmaterials to address the fifth and final barrier—the efficient andtimely separation of polymer from DNA in the nucleus—has not beenadequately addressed.

This “ultimate” barrier to efficient transfection presents a challengingproblem from a design perspective, as designing methods to surmount itcan introduce a functionality that is contrary to that required forefficient DNA condensation (i.e., barrier 1). Kircheis, R.; Wightman,L.; Wagner, E. (2001) Design and Gene Delivery Activity of ModifiedPolyethyleneimines. Advanced Drug Delivery Reviews, 53, 341-358.Cationic polymers spontaneously self-assemble with anionic DNA throughelectrostatic interactions to form condensed interpolyelectrolytecomplexes—a process that is driven entropically by the elimination ofsmall salts (e.g., NaCl) formed upon complex formation. Kabanov, A. V.;Feigner, P. L.; Seymour, L. W., in Self-Assembling Complexes for GeneDelivery: From Laboratory to Clinical Trial, John Wiley and Sons, NewYork, 1998.

Cationic polymers undergo self-assembly with anionic plasmid DNA to formcondensed complexes. The reverse of this process—the intracellulardissociation of DNA from condensed interpolyelectrolytecomplexes—appears to be unfavorable under physiological conditions andpresents a substantial obstacle to efficient gene delivery.

Although the effects of pH, temperature, salt concentration, andmolecular weight on the dissociation of model interpolyelectrolytecomplexes are generally well understood, the mechanisms through whichdissociation occurs for polymer complexes in the cytoplasm or nucleus ofa cell are currently unclear. Bronich, T. K.; Nguyen, H. K.; Eisenberg,A.; Kabanov, A. V. (2000) Recognition of DNA Topology in ReactionsBetween Plasmid DNA and Cationic Polymers. J. Am. Chem. Soc., 122,8339-8343. That meaningful levels of transfection are observed usingpolymeric vectors suggests that dissociation does occur, presumablymediated by ion exchange with other intracellular polyelectrolytes.However, recent analytical experiments suggest that DNA/polycationcomplexes are stable toward intracellular dissociation and that theinefficiency of this “unpackaging” process presents a substantialphysical barrier to transfection. Godbey, W. T.; Wu, K.; Mikos, A. G.(1999) Tracking the Intracellular Path of Poly(ethyleneimine)/DNAComplexes for Gene Delivery. Proc. Natl. Acad. Sci. USA, 96, 5177-5181;Schaffer, D. V.; Fidelman, N. A.; Dan, N.; Lauffenburger, D. A. (2000)Vector Unpackaging as a Potential Barrier for Receptor-Mediated PolyplexGene Delivery. Biotechnol. Bioeng., 67, 598-606.

SUMMARY OF THE INVENTION

The present invention provides a dynamic charge state cationic polymer,or more simply a polymer, that includes a polymeric backbone formed frommonomeric units. One or more removable functional group is/are attachedto the polymeric backbone. The dynamic charge state cationic polymer hasa cationic charge density which is a characteristic of the polymericbackbone and the functional group attached to the polymeric backbone.The cationic charge density of the dynamic charge state cationic polymerdecreases when the one or more of the removable functional group(s)is/are removed from the dynamic charge state cationic polymer. Thepresent polymers can also be part of a copolymer where only one segmentof the copolymer is the dynamic charge state cationic polymer. In someembodiments, the polymeric backbone comprises a polyamine, such aspolyethyleneimine, polylysine, polyomithine or poly/lysine/ornithine. Insome embodiments, the polymers contain side chains that have primary,secondary or tertiary amines. Other examples of suitable polymericbackbones include poly(propylene imine), poly(allyl amine), poly(vinylamine), poly(amidoamine) (PAMAM), and dendrimers that are functionalizedwith terminal amine groups. Further examples include acrylate ormethacrylate polymers such as poly(2-aminoethyl methacrylate), and thelike. In some embodiments where amine functional groups are present inthe polymer, primary amines may be functionalized either once or twiceto provide a polymer that has a net negative charge once removal of theone or more removable functional group is complete. In the presentpolymers, the polymeric backbone can be linear, branched orhyperbranched, particularly when the backbone is polyethyleneimine.

In some embodiments, at least one of the one or more removablefunctional group(s) is a hydrolyzable group, such as a pendant ester.The one or more removable functional group(s) may also include a labilelinkage, such as an ester, an anhydride, an orthoester, a phosphoester,an acetal, or an amide. In certain embodiments, the polymer has theformula:

When the polymer has the formula shown above, n may be an integerranging from 5 to 100,000, x is an integer, and the mole percent of yranges from 10 percent to 100 percent based on the total of x and y. Inthe present compounds, the identity of R is not particularly limited.For example, R can be an alkyl, alkenyl, alkynyl, cycloalkyl,heterocyclic, aryl, or a heteroaryl group. R may also becarbon-containing, heteroatom-containing (N, S, O, P, etc.), linear,branched, an amino, an alkylamino, a dialkylamino, a trialkylamino,aryl, a heterocyclyl, a cyano, an amide, a carbamoyl, or the like. Whenthe R group is alkyl, R can be methyl, ethyl, propyl, butyl, pentyl,hexyl, or combinations thereof.

In some embodiments, the polymers of the invention are biodegradable andbiocompatible.

Other examples of polymers include compounds of the following formula:

In the above polymers, n may be an integer of from 5 to 100,000, A and Bare linkers which may be the same or different and can be anysubstituted or unsubstituted, branched or unbranched chain of carbonatoms or heteroatoms; R₁ may be a linker group or a covalent bond; X maybe the same or different and can be a labile linkage, which, in someinstances is negatively charged after cleavage; Y can be a linkage thatis generally not as labile as X; R₂ through R₇ and R₁₂ through R₂₀ canhave the value for R listed above and can be the same or different; R₈,R₉, R₁₀ and R₁₁ are linkers or covalent bonds; and Z can be a covalentbond or a degradable linkage. In some embodiments X is an ester linkageand Y is an amide linkage, such as NR₁₇. In some embodiments, R₁₆ isNR₂₁R₂₂, R₁₇ is H, and R₁₈ is NR₂₃R₂₄. In some embodiments, R₁₉ isNR₂₅R₂₆ and R₂₀ is NR₂₇R₂₈. R₂₁ through R₂₈ can have the value for Rlisted above and can be the same or different. When Z is a covalentbond, the polymer backbone is non-degradable. The linkers A and B can belinkers that contain carbon atoms or heteroatoms (e.g., nitrogen,oxygen, sulfur, etc.). Typically, these linkers are 1 to 30 atoms long,more preferably 1 to 15 atoms long. The linkers may be substituted withvarious substituents including, but not limited to, hydrogen atoms, andalkyl, alkenyl, alkynyl, amino, alkylamino, dialkylamino, trialkylamino,hydroxyl, alkoxy, halogen, aryl, heterocyclic, aromatic heterocyclic,cyano, amide, carbamoyl, carboxylic acid, ester, thioether,alkylthioether, thiol, and ureido groups. As would be appreciated by oneof skill in this art, each of these groups may in turn be substituted.For some polymers the ester bond will generally be readily hydrolyzablewhereas the amide bond is not readily hydrolyzable. This configurationallows more control over the change of cationic charge density of thepolymer by altering the ratio of ester bonds and amide bonds present inthe one or more removable functional group.

In the present polymers, the mole percent of the monomers comprising thepolymeric backbone substituted with the one or more removable functionalgroup range from 10 to 100 percent or from about 10 percent to about 100percent. In additional embodiments, the mole percent of the monomersattached to the removable functional group may range from about 30percent to 100 percent, 50 percent to 100 percent or 70 percent to 100percent. The polymers of the present invention may have any desiredmolecular weight, such as from 1,000 to 100,000 grams/mole, or fromabout 2,000 to 50,000 grams/mole. The dynamic charge state cationicpolymer can be associated with a ligand facilitating the delivery of thepolymer to a specific target, such as a target cell. The presentpolymers can also be part of a copolymer, which can be composed of anyother polymers, for example a polymer such as PEG or PEO which arecommonly used to give stability toward protein adsorption. The presentpolymer is generally cationic, but different functional groups attachedto the polymer can render the polymer zwitterionic. To impart a cationiccharge to the polymer, the polymeric backbone or the attached functionalgroups can be positively charged. The present polymer may also becapable of buffering changes in pH which results from the make-up of thepolymer backbone and/or the attached functional groups.

The present dynamic charge state cationic polymers may benon-immunogenic, non-toxic or both non-immunogenic and non-toxic. In thepresent polymers, the polymeric backbone can be degradable ornondegradable. The present polymers do not require that the degradationof the backbone occur at the same time as the shift in cationic charge.One skilled in the art will recognize that the measure of degradabilitywill be commensurate with the environmental conditions and desiredproperties for any particular application for the present polymers. Asone non-limiting example, for biomedical uses of the present polymers,the present invention contemplates polymers that degrade in a desiredtime frame (from an hour to a week to a month to a year) underphysiological conditions typically found in the body or in a cell orcell compartment [e.g., pH ranges from about 5.0 (endosomal/lysosomal)to 7.4 (extracellular and cytosol), a temperature of about 37° C. and anionic strength of a typical physiological solution (generally around130-150 mM NaCl, for example)]. In the present invention, thedegradability of the polymer can be measured by a variety of methods,including, but not limited to, GPC (gel permeation chromatography).

The present invention also provides the present polymers complexed withone or more anionic molecules thereby forming an interpolyelectrolytecomplex. Suitable anionic molecules may be naturally occurring,synthetic, or both. In some embodiments, suitable examples of anionicmolecules include nucleic acids, such as RNA, DNA, and analogs thereof.In other embodiments, the anionic molecule is a synthetic polyanion. Instill other embodiments, the polymers of the invention are complexedwith an anionic molecule such as nucleic acids, such as RNA, DNA, oranalogs thereof, and a synthetic polyanion. When the anionic molecule isa nucleic acid, the nucleic acid can have the sequence of a nucleic acidmolecule of interest or its complement. As such, the nucleic acid canencode for a protein or a functional fragment thereof or be useful inantisense treatment or RNA interference. In some embodiments, thenucleic acid is a plasmid.

In other embodiments, the anionic molecule or agent may be a therapeuticmolecule, diagnostic molecule, peptide, or carbohydrate, for example amacromolecular carbohydrate such as heparin.

The interpolyelectrolyte complex may have any desired size dependingupon the intended use of the interpolyelectrolyte complex. For example,when the interpolyelectrolyte complex is used for nucleic acid deliveryto a cell, the interpolyelectrolyte complex can be 50 nm to about 400nm, or from about 50 to about 250 nanometers, in size. In otherembodiments, the interpolyelectrolyte complex may be provided in alayered complex made up of one or more layers of the dynamic chargestate cationic polymer and one or more layers of the anionic molecule.

In some embodiments, the present polymer or interpolyelectrolyte complexmay be provided in a biologically compatible solution or a biologicalsolution. Further, the polymer may be provided with a pharmaceuticallyacceptable excipient or another completely different polymer (e.g.,another cationic polymer) which could be an “excipient” or could have anadded function. Accordingly, the present compounds includepharmaceutical compositions that include any of the polymers or mixturesdescribed herein.

The present invention also provides methods for delivering an anioniccompound to a cell or tissue. The present methods involve contacting acomposition that includes a present interpolyelectrolyte complex with atarget cell thereby allowing the target cell to uptake the composition.The polymer of the present invention is designed such that when theinterpolyelectrolyte complex enters the target cell, one or more of theremovable functional group(s) is/are removed from the dynamic chargestate cationic polymer which decreases the cationic charge density ofthe dynamic charge state cationic polymer. The decrease in the cationiccharge density of the polymer may be caused by the introduction ofanionic charges which promotes dissociation of the interpolyelectrolytecomplex into the dynamic charge state cationic polymer and the anionicmolecule allowing for delivery of the anionic molecule to the targetcell or cell compartment, such as an endosome, cytosol or nucleus of thecell. In some methods, at least one of the one or more of the removablefunctional group(s) is removed from the dynamic charge state cationicpolymer in a nucleus, endosome or cytosol of the target cell. In thismanner, the interpolyelectrolyte complex may dissociate primarily in thedesired compartment of the target cell and deliver the anionic moleculeto the target cell compartment. The present methods may also involveproviding the interpolyelectrolyte complex and/or preparing theinterpolyelectrolyte complex. Generally, the interpolyelectrolytecomplex will be prepared by mixing the dynamic charge state cationicpolymer with the anionic molecule thereby allowing formation of theinterpolyelectrolyte complex. In the methods where the anionic moleculeis DNA, the DNA may be delivered to the nucleus of the cell so that itis stably incorporated into the genome of the target cell. In otherembodiments, the DNA is not stably incorporated into the genome of thetarget cell.

In the present methods, the target cell or tissue can be in vitro or invivo. Where the target cell or tissue is in vivo, theinterpolyelectrolyte complex may be administered to a mammal. In someembodiments of the present methods, the cell is a eukaryotic cell.

In the present methods and polymers, removal of the one or more of theremovable functional group(s) from the dynamic charge state cationicpolymer may be at least partially hydrolytic, partially enzymatic and/orpartially photolytic removal. The present polymers and methods may alsobe designed so that the removal of the one or more of the removablefunctional group(s) from the dynamic charge state cationic polymeroccurs at a substantially constant rate or does not occur at a constantrate.

The present invention also provides kits containing the presentpolymers.

The invention also provides methods of preparing the polymers andmethods of preparing microspheres and other pharmaceutical compositionscontaining the polymers.

In yet another aspect of the invention, the polymers are used to formnanometer-scale complexes with nucleic acids. The polynucleotide/polymercomplexes may be formed by adding a solution of polynucleotide to avortexing solution of the polymer at a desired DNA/polymerconcentration. The weight to weight ratio of polynucleotide to polymermay range from 1:0.1 to 1:50, preferably from 1:1 to 1:20, morepreferably from 1:1 to 1:10. The cationic polymers condense thepolynucleotide into soluble particles typically 50-500 nm in size. Thesepolynucleotide/polymer complexes may be used in the delivery ofpolynucleotides to cells. In some embodiments, these complexes arecombined with pharmaceutical excipients to form pharmaceuticalcompositions suitable for delivery to animals including humans.

In another aspect of the invention, the polymers are used to encapsulatetherapeutic, diagnostic, and/or prophylactic agents includingpolynucleotides to form microparticles. Typically these microparticlesare one or more orders of magnitude larger than thepolynucleotide/polymer complexes. The microparticles range from 1micrometer to 500 micrometers. In some such embodiments, themicroparticles allow for the delivery of labile small molecules,proteins, peptides, and/or polynucleotides to an individual. Themicroparticles may be prepared using any of the techniques known in theart to make microparticles, such as, for example, double emulsion andspray drying. In some embodiments, the microparticles may be used forpH-triggered delivery of the encapsulated contents due to thepH-responsive nature of the polymers (i.e., being more soluble at lowerpH).

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1-11 show the visualization of polycation/DNA interpolyelectrolytecomplex formation and subsequent release of DNA from destabilizedinterpolyelectrolyte complexes of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention focuses on providing polymers that address thefinal physical barrier to efficient delivery—the timely intracellulardissociation of DNA from polymer/DNA interpolyelectrolyte complexes. Thegeneral approach of the present invention is based on the synthesis ofcationic polymers that undergo dynamic changes in charge states (i.e.,from cationic to “less cationic” or zwitterionic) to trigger the“unpackaging” of anionic molecules, such as DNA, frominterpolyelectrolyte complexes. In one embodiment, the synthetic designis based on the introduction of side-chain esters to linearpoly(ethylene imine) via conjugate addition chemistry. Without limitingthe scope of the invention, it is believed that the dynamic introductionof carboxylate groups into these polymers, which occurs via the gradualhydrolysis of pendant ester groups, effectively lowers the cationiccharge densities of the polymers and promotes the dissociation ofpolymer/anion complexes. The disclosed polymers undergo a shift incharged states as a function of time to initiate the efficient andtimely unpackaging of DNA from condensed particles in the intracellularenvironment.

Affecting the timely intracellular dissociation of polycation/anioniccomplexes is an important and unsolved problem. The present polymersaddress this final physical barrier to anion delivery and can lead toeffective new formulations for anion delivery and, importantly, allowexisting solutions to earlier barriers to be exploited more fully. Priorresearch has generally approached this problem through the synthesis ofhydrolytically degradable polycations or by the development ofdisulfide-crosslinked networks that use exposure to reductiveintracellular environments to trigger degradation and enhance polymerdissociation. Peterson, H.; Merdan, T.; Kunath, K.; Fischer, D.; Kissel,T. (2002) Poly(ethyleneimine-co-L-lactamide-co-succinamide): ABiodegradable Polyethyleneimine Derivative with an AdvantageouspH-Dependent Hydrolytic Degradation for Gene Delivery. BioconjugateChem., 13, 812-821; Wang, J.; Mao, H.; Leong, K. W. (2001) A NovelBiodegradable Gene Carrier Based on Polyphosphoester. J. Am. Chem. Soc.,123, 9480-9481; Lim, Y.; Kim, C.; Kim, K.; Kim, S. W.; Park, J. (2000)Development of a Safe Gene Delivery System Using Biodegradable Polymer,Poly[α-(4-aminobutyl)-L-glycolic acid]. J. Am. Chem. Soc., 122,6524-6525; Lynn, D. M.; Langer, R. (2000) Degradable Poly(β-AminoEsters): Synthesis, Characterization, and Self-Assembly with Plasmid DNAJ. Am. Chem. Soc., 122, 10761-10768; Putnam, D.; Langer, R. (1999)Poly(4-hydroxy-1-proline ester): Low-Temperature Polycondensation andPlasmid DNA Complexation. Macromolecules, 32, 3658-3662; Oupicky, D.;Parker, A. L.; Seymour, L. W. (2002) Laterally Stabilized Complexes ofDNA with Linear Reducible Polycations: Strategy for TriggeredIntracellular Activation of DNA Delivery Vectors. J. Am. Chem. Soc.,124, 8-9; Pichon, C.; LeCam, E.; Guérin, B.; Coulaud, D.; Delain, E.;Midoux, P. (2002) Poly[Lys-(AEDTP)]: A Cationic Polymer That AllowsDissociation of pDNA/Cationic Polymer Complexes in a Reductive Mediumand Enhances Polyfection. Bioconjugate Chem., 13, 76-82; Gosselin, M.A.; Guo, W.; Lee, R. J. (2001) Efficient Gene Transfer Using ReversiblyCross-Linked Low Molecular Weight Polyethyleneimine. Bioconjugate Chem.,12, 989-994. The present invention provides a different approach, basedon synthetic polymers that shift charge states dynamically to triggerthe “unpackaging” of DNA from interpolyelectrolyte complexes through theintroduction of repulsive electrostatic interactions. One embodiment isoutlined below:

The above scheme demonstrates the general concept of dynamicintroduction of carboxylate groups to a cationic poly(amine) viaside-chain ester hydrolysis. Assuming full protonation for illustrativepurposes, the overall positive charge on polymer 1 is the sum of ‘n+m’,and this polymer is capable of forming interpolyelectrolyte complexeswith polyanions such as DNA. Hydrolysis of the ester side chains inpolymer 1 (assuming complete hydrolysis of all ester side chains)introduces ‘m’ negative carboxylate groups and thus reduces the overallpositive charge on polymer 2 to ‘n’, promoting dissociation of polymercomplexes.

In this embodiment, the dynamic introduction of carboxylate groups intocationic polymers (via the gradual hydrolysis of pendant ester groups)effectively “neutralizes” or reduces the charge densities of thesepolymers and exerts a destabilizing influence that can promote thedissociation of polymer/DNA complexes. For example, the overall positivecharge on polymer 1 is reduced from (n+m)+ to (n)+ upon hydrolysis(above scheme, assuming full protonation for illustrative purposes andcomplete hydrolysis of ester groups in polymer 2). One skilled in theart will recognize that this charge shifting capability can be readilyapplied to other polymers.

In some embodiments, the present polymers are based on linearpoly(ethylene imine) (PEI) as the structural template from whichpolymers are synthesized. Linear PEI is an attractive template forseveral reasons: 1) it is commercially available or can be synthesizeddirectly via the ring-opening polymerization of substituted oxazolines,2) it is a proven gene transfer agent already capable of surmountingmany “early” barriers to transfection, and 3) the linear array ofsecondary amines facilitates the introduction of new functionalitythrough well defined chemistry. Odian, G., Principles of Polymerization,John Wiley and Sons, Inc., New York, 1991; Kircheis, R.; Wightman, L.;Wagner, E. (2001) Design and Gene Delivery Activity of ModifiedPolyethyleneimines. Advanced Drug Delivery Reviews, 53, 341-358; Yin,R.; Zhu, Y.; Tomalia, D. A. (1998) Architectural Copolymers: Rod-Shaped,Cylindrical Dendrimers. J. Am. Chem. Soc., 120, 2678-2679. The methodsbelow can also be applied to branched or hyperbranched PEI (also aneffective gene transfer agent). Boussif, O.; Lezoualc'H, F.; Zanta, M.A.; Mergny, M. D.; Scherman, D.; Demeneix, B.; Behr, J. P. (1995) AVersatile Vector for Gene and Oligonucleotide Transfer Into Cells inCulture and In Vivo—Polyethyleneimine Proc. Natl. Acad. Sci. USA, 92,7297-7301; Kircheis, R.; Wightman, L.; Wagner, E. (2001) Design and GeneDelivery Activity of Modified Polyethyleneimines. Advanced Drug DeliveryReviews, 53, 341-358. In some embodiments, the present inventionmodifies polymers that are already known as effective polyanion deliveryagents.

In some embodiments, the synthetic strategy is based on the conjugateaddition of the secondary amines of PEI to acrylate compounds. Theexhaustive functionalization of linear PEI has been demonstrated in thecontext of dendrimer synthesis using methyl acrylate, yieldingester-functionalized polymer 1 (R=methyl, n=0) having regularlyrepeating tertiary amines in the polymer backbone. Yin, R.; Zhu, Y.;Tomalia, D. A. (1998) Architectural Copolymers: Rod-Shaped, CylindricalDendrimers. J. Am. Chem. Soc., 120, 2678-2679.

The introduction of the alkyl ester groups in some of the presentpolymers may influence the pKa's and increase the steric bulksurrounding the amines, and may consequently affect the ability of thepolymers to form complexes with DNA. To investigate the relationshipsbetween charge density, backbone substitution, and interpolyelectrolytecomplex formation (methods described below) a family of polymers with arange of mole percent substitution (i.e., from about 10% to 100%functionalized) have been synthesized. Putnam, D.; Gentry, C. A.; Pack,D. W.; Langer, R. (2001) Polymer-Based Gene Delivery with LowCytotoxicity by a Unique Balance of Side-Chain Termini. Proc. Natl.Acad. Sci. USA, 98, 1200-1205; Jeong, J. H.; Song, S. H.; Lim, D. W.;Lee, H.; Park, T. G. (2001) DNA Transfection Using LinearPoly(ethyleneimine) Prepared by Controlled Acid Hydrolysis ofPoly(2-ethyl-2-oxazoline). J. Control. Release, 73, 391-399. Similarly,polymers with varying hydrophobicities and charge densities have beensynthesized by conjugate addition to more hydrophobic acrylates (i.e.,where R=ethyl, propyl, butyl, or combinations thereof). Because bothtransfection and the “unpackaging” of DNA from interpolyelectrolytecomplexes appear to be related to polycation molecular weight (withshorter polymers tending to dissociate more readily than largerpolymers), individual syntheses were conducted using PEI of differentmolecular weights and polydispersities to explore these relationshipsand optimize polymer behavior. Godbey, W. T.; Wu, K. K.; Mikos, A. G.(1999) Size Matters: Molecular Weight Affects the Efficiency ofPoly(ethyleneimine) as a Gene Delivery Vehicle. J. Biomed. Mater. Res.,45, 268-275; Schaffer, D. V.; Fidelman, N. A.; Dan, N.; Lauffenburger,D. A. (2000) Vector Unpackaging as a Potential Barrier forReceptor-Mediated Polyplex Gene Delivery. Biotechnol. Bioeng., 67,598-606.

As described above, polycations and plasmid DNA spontaneouslyself-assemble under physiological conditions to form nanometer-scaleinterpolyelectrolyte complexes. These complexes may be destabilized byexposure to high salt concentrations or by the presence of otherpolyelectrolytes that initiate polyion exchange processes. Bronich, T.K.; Nguyen, H. K.; Eisenberg, A.; Kabanov, A. V. (2000) Recognition ofDNA Topology in Reactions Between Plasmid DNA and Cationic Polymers. J.Am. Chem. Soc., 122, 8339-8343. Agarose gel electrophoresis provides forconvenient visualization of polycation/DNA interpolyelectrolyte complexformation and is used as a qualitative measure for the subsequentrelease of DNA from destabilized complexes. Lynn, D. M.; Langer, R.(2000) Degradable Poly(β-Amino Esters): Synthesis, Characterization, andSelf-Assembly with Plasmid DNA J. Am. Chem. Soc., 122, 10761-10768;Putnam, D.; Langer, R. (1999) Poly(4-hydroxy-1-proline ester):Low-Temperature Polycondensation and Plasmid DNA Complexation.Macromolecules, 32, 3658-3662; Wang, J.; Mao, H.; Leong, K. W. (2001) ANovel Biodegradable Gene Carrier Based on Polyphosphoester. J. Am. Chem.Soc., 123, 9480-9481. To obtain more quantitative information about thebiophysical interactions between DNA and the polymers synthesized above,one may also use an established fluorescence-based ethidium bromideexclusion assay to investigate the kinetics of complex destabilization.Bronich, T. K.; Nguyen, H. K.; Eisenberg, A.; Kabanov, A. V. (2000)Recognition of DNA Topology in Reactions Between Plasmid DNA andCationic Polymers. J. Am. Chem. Soc., 122, 8339-8343.

The kinetics of ester hydrolysis and interpolyelectrolyte complexdisruption of some of the present polymers was evaluated over a range ofpH, temperature, and salt concentration, focusing on those conditionslikely to be encountered by these materials during transfection (e.g.,37° C., 150 mM NaCl, and pH values ranging from 5.1 to 7.2 toapproximate the pH within endosomal vesicles and the cytoplasm,respectively). Interpolyelectrolyte complexes were formed at variouspolymer/DNA ratios by standard mixing protocols. Dynamic lightscattering (DLS) was used to determine the size distributions of thecomplexes and the relationships between backbone substitution, cationcharge density, side-chain hydrolysis, and interpolyelectrolyte complexparticle charge. Zeta-potential analysis may also be used tocharacterize the polymer/DNA complexes. Bronich, T. K.; Nguyen, H. K.;Eisenberg, A.; Kabanov, A. V. (2000) Recognition of DNA Topology inReactions Between Plasmid DNA and Cationic Polymers. J. Am. Chem. Soc.,122, 8339-8343; Putnam, D.; Gentry, C. A.; Pack, D. W.; Langer, R.(2001) Polymer-Based Gene Delivery with Low Cytotoxicity by a UniqueBalance of Side-Chain Termini. Proc. Natl. Acad. Sci. USA, 98,1200-1205; Lynn, D. M.; Langer, R. (2000) Degradable Poly(β-AminoEsters): Synthesis, Characterization, and Self-Assembly with Plasmid DNAJ. Am. Chem. Soc., 122, 10761-10768; Gonzalez, H.; Hwang, S. J.; Davis,M. E. (1999) New Class of Polymers for the Delivery of MacromolecularTherapeutics. Bioconjugate Chem., 10, 1068-1074.

The present polymers have been rationally designed to promote theintracellular dissociation of DNA from interpolyelectrolyte complexes.The functional polymers may change charge states as a function of time,allowing the initial formation of polymer DNA complexes (to addressearly barriers to transfection) and facilitating intracellulardissociation through the introduction of repulsive electrostaticinteractions (to address late barriers). An appropriate balance ofcharge density and side-chain hydrolysis allows these competing factorsto be addressed on a time scale relevant to biological aniondelivery/gene transfer and expression. The present polymers may also bedesigned to have a desired rate of hydrolysis so that the charge shiftof the polymer occurs on the desired time scale.

The polymers may be used in the pharmaceutical/drug delivery arts todeliver polynucleotides, proteins, small molecules, peptides, antigen,drugs, or the like to a patient, tissue, organ, cell, or the like.

The polymers may also be used to complex polynucleotides and therebyenhance the delivery of polynucleotide and prevent their degradation.The polymers may also be used in the formation of nanoparticles ormicroparticles containing encapsulated agents. Due to some of thepolymers' properties of being biocompatible and biodegradable, theseformed particles are also biodegradable and biocompatible and may beused to provide controlled, sustained release of the encapsulated agent.These particles may also be responsive to pH changes.

Polymers

The present polymers are dynamic charge state cationic polymers thathave a cationic charge density which is a characteristic of thepolymeric backbone and the functional groups attached to the polymericbackbone. The polymers are designed such that the cationic chargedensity of the dynamic charge state cationic polymer decreases when oneor more of the removable functional group(s) is removed from the dynamiccharge state cationic polymer.

Based on these criteria, the polymer backbone is not particularlylimited. In some embodiments, the polymer backbone is positivelycharged, whereas in others the polymeric backbone is neutral. Generallyspeaking, the charge of the polymer backbone is measured underphysiological conditions, such as at physiological pH. In some instancesthe polymer backbone is made up of repeating units of polyamines, suchas polyethyleneimine, polylysine, polyornithine orpoly/lysine/ornithine, because such polymers provide the desiredcationic charge density and are easy to manipulate. Specific examples ofsuch suitable polymeric backbones include poly(propylene imine),poly(allyl amine), poly(vinyl amine), poly(amidoamine) (PAMAM), anddendrimers that are functionalized with terminal amine groups. Furtherexamples of polymeric backbones suitable for use in the presentinvention include acrylate or methacrylate polymers such aspoly(2-aminoethyl methacrylate), and the like. In some embodiments,where amine functional groups are present in the polymer, primary aminesmay be functionalized either once or twice to provide a polymer that hasa net negative charge once removal of the one or more removablefunctional group(s) is complete. In the present polymers, the polymericbackbone may be linear, branched or hyperbranched.

The present polymer is generally cationic, but different functionalgroups attached to the polymer can render the polymer zwitterionic. Thepresent polymer may also be capable of buffering changes in pH whichresults from the make-up of the polymer backbone and/or the attachedfunctional groups.

Similar to the backbone, the identity of the one or more removablefunctional group(s) of the present polymers is not particularly limitedas long as removal of the one or more removable functional group(s)decreases the cationic charge density of the polymer. As used herein,“removable functional group” means a chemical group that, upon removal,will decrease the cationic charge density of the polymer. As will beapparent to the skilled artisan, polymers whose cationic charge densitydecreases in this manner can have a variety of features. For example,the removable functional group may be positively charged so that removalof the removable functional group reduces cationic charge density. Thismay be particularly important where the polymeric backbone is notpositively charged. In other embodiments the removable functional groupmay be positively charged or neutral prior to removal, but provide anegatively charged species after it is removed from the polymer. Oneexample of such a scheme is provided when the removable group contains ahydrolyzable ester. Other configurations that achieve the chargeshifting properties of the present polymers will be apparent to thoseskilled in the art. When removable functional groups provide anegatively charged species after removal from the polymer backbone andthe backbone itself is neutral, then the present polymers can shift frombeing cationic to anionic when the removable functional group isremoved.

Examples of removable functional groups suitable for use in the presentpolymers include side chains that have primary, secondary or tertiaryamines. Primary amines useful in the present polymers include, but arenot limited to, methylamine, ethylamine, isopropylamine, aniline,substituted anilines, and ethanolamine. In some embodiments, at leastone of the one or more removable functional group(s) is a hydrolyzablegroup, such as a pendant ester. Specific examples for the one or moreremovable functional group(s) may also include a labile linkage, such asan ester, an anhydride, an orthoester, a phosphoester, an acetal, or anamide.

The present polymer is generally cationic, but different functionalgroups attached to the polymer may render the polymer zwitterionic. Thepresent polymer may, also be capable of buffering changes in pH whichresults from the make-up of the polymer backbone and/or the attachedfunctional groups.

More specifically, polymers having the following structure are suitablefor use in the present invention:

When the polymer has the above formula, n is an integer ranging from 5to 100,000, x is an integer, and the mole percent of y may range from 10percent to 100 percent (based on the total of x and y). In the presentcompounds, the identity of R is not particularly limited. For example, Rcan be an alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclic, aryl, orheteroaryl group. R may also be carbon-containing, heteroatom containing(N, S, O, P, etc), linear, branched, an amino, an alkylamino, adialkylamino, a trialkylamino, an aryl, a heterocyclic, a cyano, anamide, a carbamoyl group, or the like. When the R group is alkyl, R canbe methyl, ethyl, propyl, butyl, pentyl, hexyl or combinations thereof.

Other examples of polymers include:

In the above polymers, n is an integer ranging from 5 to 100,000, A andB are linkers which may be the same or different and may be anysubstituted or unsubstituted, branched or unbranched chain of carbonatoms or heteroatoms; R₁ is a linker group or a covalent bond; X is thesame or different and is a labile linkage, which, in some instances, isnegatively charged after cleavage; Y is a linkage that is generally notas labile as X; R₂ through R₇ and R₁₂ through R²⁰ have the value for Rlisted above and are the same or different; R₈, R₉, R₁₀ and R₁₁ arelinkers or covalent bonds; and Z is a covalent bond or a degradablelinkage. In some embodiments, X is an ester linkage and Y is an amidelinkage, such as NR₁₇. In some embodiments, R₁₆ is NR₂₁R₂₂, R₁₇ is H,and R₁₈ is NR₂₃R₂₄. In some embodiments, R₁₉ is NR₂₅R₂₆, and R₂₀ isNR₂₇R₂₈. R₂₁ through R₂₈ may have the value for R listed above and arethe same or different. When Z is a covalent bond, the polymer backboneis non-degradable. The linkers A and B may be linkers that includecarbon atoms or heteroatoms (e.g., nitrogen, oxygen, sulfur, etc.).Typically, such linkers are 1 to 30 atoms long, or in some case are 1 to15 atoms long. The linkers may be substituted with various substituentsincluding, but not limited to, hydrogen atoms, and alkyl, alkenyl,alkynyl, amino, alkylamino, dialkylamino, trialkylamino, hydroxyl,alkoxy, halogen, aryl, heterocyclic, aromatic heterocyclic, cyano,amide, carbamoyl, carboxylic acid, ester, thioether, alkylthioether,thiol, and ureido groups. As would be appreciated by one of skill in theart, each of these groups may in turn be substituted. For some polymers,the ester bond will generally be readily hydrolyzable whereas the amidebond is not readily hydrolyzable. This configuration allows more controlover the change of cationic charge density of the polymer by alteringthe ratio of ester bonds and amide bonds present in the removablefunctional group(s).

In the present polymers, the mole percent of the monomers comprising thepolymeric backbone substituted with the one or more removable functionalgroup(s) range from about 10 percent to about 100 percent or from 10percent to 100 percent. In additional embodiments, the mole percent ofthe monomers attached to the one or more removable functional group mayrange from about 30 percent to 100 percent, from 50 percent to 100percent, or from 70 percent to 100 percent. The polymers of the presentinvention may have any desired molecular weight, such as from 1,000 to100,000 grams/mole, or from about 2,000 to 50,000 grams/mole in someembodiments.

The present dynamic charge state cationic polymers can benon-immunogenic, non-toxic or both non-immunogenic and non-toxic. In thepresent polymers, polymeric backbone can be degradable or nondegradable.In some embodiments, the polymers of the invention are biodegradable andbiocompatible.

The molecular weights of the polymers may range from 5,000 g/mol to over100,000 g/mol in some embodiments and from 4,000 g/mol to 50,000 g/molin other embodiments. In some embodiments, the polymers are relativelynon-cytotoxic. In other embodiments, the polymers are biocompatible andbiodegradable. In one embodiment, the polymers of the present inventionhave pK_(a)s ranging from 5.5 to 7.5 or, in some embodiments, from 6.0and 7.0. In other embodiments, the polymer may be designed to have adesired pK_(a) ranging from 3.0 to 9.0, and in some embodiments from 5.0to 8.0. The polymers are particularly attractive for drug delivery forseveral reasons including the following: 1) they may contain aminogroups for interacting with DNA and other negatively charged agents, forbuffering the pH, for causing endosomolysis, etc.; 2) they may containdegradable polyester linkages; 3) they may be synthesized fromcommercially available starting materials; and 4) they may be pHresponsive and engineered to have a desired pK_(a).

As described herein, any chemical group that satisfies the valency ofeach atom may be substituted for any hydrogen atom.

Synthesis of Polymers

The polymers may be prepared by any method known in the art. In someembodiments, the polymers are prepared from commercially availablestarting materials. In other embodiments, the polymers are prepared fromeasily and/or inexpensively prepared starting materials.

In some embodiments, the polymer is prepared via conjugate addition ofmethyl acrylate to linear poly(ethylene imine). Examples of thissynthetic scheme are shown in the examples.

The synthesized polymer may be purified by any technique known in theart including, but not limited to, precipitation, crystallization,chromatography, etc. In some embodiments, the polymer need not bepurified. In some embodiments, the polymer is purified through repeatedprecipitations from an organic solvent (e.g., diethyl ether, hexane,etc.). In some embodiments, the polymer is isolated as a salt, such as ahydrochloride salt or a pharmaceutically acceptable salt. As would beappreciated by one of skill in this art, the molecular weight of thesynthesized polymer and the extent of cross-linking may be determined bythe reaction conditions (e.g., temperature, starting materials,concentration, order of addition, solvent, etc.) used in the synthesis(Odian Principles of Polymerization 3rd Ed., New York: John Wiley &Sons, 1991; Stevens Polymer Chemistry: An Introduction 2nd Ed., NewYork: Oxford University Press, 1990; each of which is incorporatedherein by reference).

In one embodiment, a library of different polymers is prepared inparallel. A different amount of the one or more removable functionalgroup is added to each vial in a set of vials used to prepare thelibrary. The array of vials is incubated at a temperature and length oftime sufficient to allow functionalization of the polymers to occur. Thepolymers may then be isolated and purified using techniques known in theart. The polymers may then be screened using high-throughput techniquesto identify polymers with a desired characteristic (e.g., solubility inwater, solubility at different pH, ability to bind polynucleotides,ability to bind heparin, ability to bind small molecules, ability toform microparticles, ability to increase transfection efficiency, etc.).In certain embodiments, the polymers may be screened for properties orcharacteristics useful in gene therapy (e.g., ability to bindpolynucleotides, increase in transfection efficiency). In otherembodiments, the polymers may be screened for properties orcharacteristics useful in the art of tissue engineering (e.g., abilityto support tissue or cell growth, ability to promote cell attachment).

Interpolyelectrolyte Complexes

The present invention also provides the present polymers complexed withone or more anions thereby forming an interpolyelectrolyte complex. Inthe interpolyelectrolyte complexes of the present invention, the anionbound by the polymer is not particularly limited. In some embodiments,the anion need only have at least two negative charges. Suitableexamples of anionic molecules include nucleic acids, proteins, peptides,carbohydrates, therapeutic molecules or agents, diagnostic molecules oragents, prophylactic agents, small molecules, organometallic compounds,drugs, vaccines, immunological agents, and the like. The anions may benaturally occurring or synthetic as synthetic polyanions may also beused to form interpolyelectrolyte complexes of the invention. In someembodiments, the polymers of the invention are complexed to an anionmolecule such as nucleic acids, such as DNA, RNA, or analogs orfragments thereof. In some such embodiments, the polymers may further becomplexed to a synthetic polyanion.

The present invention also provides arrays of interpolyelectrolytecomplexes arranged on a suitable surface. The arrays can haveinterpolyelectrolyte complexes that include different combinations ofpolymers and/or anions at discrete and defined positions. Theinterpolyelectrolyte arrays can be used for a variety of high-throughputtesting procedures, such as drug discovery, cell transfection, and thelike. In these methods, cells will be placed, plated and/or cultured onthe interpolyelectrolyte array and analyzed. Suitable examples of arrayconfigurations and methods for producing and using the presentinterpolyelectrolyte complex arrays are discussed in U.S. Pat. No.6,544,790 which is incorporated herein by reference in its entirety andfor all purposes as if fully set forth herein.

Polynucleotide Complexes

The ability of cationic compounds to interact with negatively chargedpolynucleotides through electrostatic interactions is well known.Cationic polymers such as poly(lysine) have been prepared and studiedfor their ability to complex polynucleotides. The interaction of thepolymer with the polynucleotide is thought to at least partially preventthe degradation of the polynucleotide. By neutralizing the charge on thebackbone of the polynucleotide, the neutral orslightly-positively-charged complex is also able to more easily enterthe cell, for example by traversing through the hydrophobic membranes(e.g., cytoplasmic, lysosomal, endosomal, nuclear) of the cell. In someembodiments, the complex is positively charged. In some embodiments, thecomplex has a positive zeta-potential, more preferably thezeta-potential is between +1 and +30.

Polynucleotides or derivatives thereof are contacted with the polymersunder conditions suitable to form polynucleotide/polymer complexes. Thepolymer is preferably at least partially protonated so as to form acomplex with the negatively charged polynucleotide. In some embodiments,the polynucleotide/polymer complexes form nanoparticles that are usefulin the delivery of polynucleotides to cells. In some such embodiments,the diameter of the nanoparticles ranges from 50-500 nm, more preferablythe diameter of the nanoparticles ranges from 50-200 nm, and mostpreferably from 90-150 nm. The nanoparticles may be associated with atargeting agent as described below.

Polynucleotide

The polynucleotide to be complexed or encapsulated by the polymers maybe any nucleic acid including but not limited to RNA and DNA. Thepolynucleotides may be of any size or sequence, and they may be single-or double-stranded. In certain embodiments, the polynucleotide isgreater than 100 base pairs long. In certain other embodiments, thepolynucleotide is greater than 1000 base pairs long and may be greaterthan 10,000 base pairs long. The polynucleotide is preferably purifiedand/or substantially pure. Preferably, the polynucleotide is greaterthan 50% pure, more preferably greater than 75% pure, and mostpreferably greater than 95% pure. The polynucleotide may be provided byany means known in the art. In certain embodiments, the polynucleotidehas been engineered using recombinant techniques (for a more detaileddescription of these techniques, please see Ausubel et al. CurrentProtocols in Molecular Biology (John Wiley & Sons, Inc., New York,1999); Molecular Cloning: A Laboratory Manual, 2nd Ed., ed. by Sambrook,Fritsch, and Maniatis (Cold Spring Harbor Laboratory Press: 1989); eachof which is incorporated herein by reference in its entirety and for allpurposes as if fully set forth herein). The polynucleotide may also beobtained from natural sources and purified from contaminating componentsfound normally in nature. The polynucleotide may also be chemicallysynthesized in a laboratory. In some embodiments, the polynucleotide issynthesized using standard solid phase chemistry.

The polynucleotide may be modified by chemical or biological means. Incertain embodiments, these modifications lead to increased stability ofthe polynucleotide. Modifications include methylation, phosphorylation,end-capping, and the like.

Derivatives of polynucleotides may also be used in the presentinvention. These derivatives include modifications in the bases, sugars,and/or phosphate linkages of the polynucleotide. Modified bases include,but are not limited to, those found in the following nucleoside analogs:2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyladenosine, 5-methylcytidine, C⁵-bromouridine, C⁵-fluorouridine,C⁵-iodouridine, C⁵-propynyl-uridine, C⁵-propynyl-cytidine,C⁵-methylcytidine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine,8-oxoguanosine, O(6)-methylguanine, and 2-thiocytidine. Modified sugarsinclude, but are not limited to, 2′-fluororibose, ribose,2′-deoxyribose, dideoxyribose, 2′,3′-dideoxyribose, arabinose (the2′-epimer of ribose), acyclic sugars, and hexoses. The nucleosides maybe strung together by linkages other than the phosphodiester linkagefound in naturally occurring DNA and RNA. Modified linkages include, butare not limited to, phosphorothioate and 5′-N-phosphoramidite linkages.Combinations of the various modifications may be used in a singlepolynucleotide. These modified polynucleotides may be provided by anymeans known in the art. However, as will be appreciated by those ofskill in this art, the modified polynucleotides are preferably preparedusing synthetic chemistry in vitro.

The polynucleotides to be delivered may be in any form. For example, thepolynucleotide may be a circular plasmid, a linearized plasmid, acosmid, a viral genome, a modified viral genome, an artificialchromosome, or the like.

The polynucleotide may be of any sequence. In certain embodiments, thepolynucleotide encodes a protein or peptide. The encoded proteins may beenzymes, structural proteins, receptors, soluble receptors, ionchannels, pharmaceutically active proteins, cytokines, interleukins,antibodies, antibody fragments, antigens, coagulation factors, albumin,growth factors, hormones, insulin, etc. The polynucleotide may alsocomprise regulatory regions to control the expression of a gene. Theseregulatory regions may include, but are not limited to, promoters,enhancer elements, repressor elements, TATA box, ribosomal bindingsites, stop site for transcription, and the like. In other embodiments,the polynucleotide is not intended to encode a protein. For example, thepolynucleotide may be used to fix an error in the genome of the cellbeing transfected.

The polynucleotide may also be provided as an antisense agent or RNAinterference (RNAi) agent (Fire et al. Nature 391:806-811, 1998;incorporated herein by reference in its entirety and for all purposes asif fully set forth herein). Antisense therapy is meant to include, e.g.,administration or in situ provision of single- or double-strandedoligonucleotides or their derivatives which specifically hybridize,e.g., bind, under cellular conditions, with cellular mRNA and/or genomicDNA, or mutants thereof, so as to inhibit expression of the encodedprotein, e.g., by inhibiting transcription and/or translation (Crooke“Molecular Mechanisms of Action of Antisense Drugs” Biochim. Biophys.Acta 1489(1):31-44, 1999; Crooke “Evaluating the Mechanism of Action ofAntiproliferative Antisense Drugs” Antisense Nucleic Acid Drug Dev.10(2):123-126, discussion 127, 2000; Methods in Enzymology volumes313-314, 1999; each of which is incorporated herein by reference in itsentirety and for all purposes as if fully set forth herein). The bindingmay be by conventional base pair complementarity, or, for example, inthe case of binding to DNA duplexes, through specific interactions inthe major groove of the double helix (i.e., triple helix formation)(Chan et al. J. Mol. Med. 75(4):267-282, 1997; incorporated herein byreference).

In certain embodiments, the polynucleotide to be delivered comprises asequence encoding an antigenic peptide or protein. Nanoparticlescontaining these polynucleotides may be delivered to an individual toinduce an immunologic response sufficient to decrease the chance of asubsequent infection and/or lessen the symptoms associated with such aninfection. The polynucleotide of these vaccines may be combined withinterleukins, interferon, cytokines, and adjuvants such as choleratoxin, KLH, alum, Freund's adjuvant, or the like. A large number ofadjuvant compounds are known. A useful compendium of many such compoundsis prepared by the National Institutes of Health and can be found on theWorld Wide Web(http:/www.niaid.nih.gov/daids/vaccine/pdf/compendium.pdf, incorporatedherein by reference; see also Allison Dev. Biol. Stand. 92:3-11, 1998;Unkeless et al. Arum. Rev. Immunol. 6:251-281, 1998; and Phillips et al.Vaccine 10:151-158, 1992, each of which is incorporated herein byreference in its entirety and for all purposes as if fully set forthherein).

The antigenic protein or peptides encoded by the polynucleotide may bederived from such bacterial organisms as Streptococccus pneumoniae,Haemophilus influenzae, Staphylococcus aureus, Streptococcus pyrogenes,Corynebacterium diphtheriae, Listeria monocytogenes, Bacillus anthracis,Clostridium tetani, Clostridium botulinum, Clostridium perfringens,Neisseria meningitidis, Neisseria gonorrhoeae, Streptococcus mutans,Pseudomonas aeruginosa, Salmonella typhi, Haemophilus parainfluenzae,Bordetella pertussis, Francisella tularensis, Yersinia pestis, Vibriocholerae, Legionella pneumophila, Mycobacterium tuberculosis,Mycobacterium leprae, Treponema pallidum, Leptospirosis interrogans,Borrelia burgdorferi, Camphylobacter jejuni, and the like; from suchviruses as smallpox, influenza A and B, respiratory syncytial virus,parainfluenza, measles, HIV, varicella-zoster, herpes simplex 1 and 2,cytomegalovirus, Epstein-Barr virus, rotavirus, rhinovirus, adenovirus,papillomavirus, poliovirus, mumps, rabies, rubella, coxsackieviruses,equine encephalitis, Japanese encephalitis, yellow fever, Rift Valleyfever, hepatitis A, B, C, D, and E virus, and the like; and from suchfungal, protozoan, and parasitic organisms such as Cryptococcusneoformans, Histoplasma capsulatum, Candida albicans, Candidatropicalis, Nocardia asteroides, Rickettsia ricketsii, Rickettsia typhi,Mycoplasma pneumoniae, Chlamydial psittaci, Chlamydial trachomatis,Plasmodium falciparum, Trypanosoma brucei, Entamoeba histolytica,Toxoplasma gondii, Trichomonas vaginalis, Schistosoma mansoni, and thelike.

Microparticles

The polymers of the present invention may also be used to form drugdelivery devices. The polymers may be used to encapsulate anioniccompounds including polynucleotides, small molecules, proteins,peptides, metals, organometallic compounds, and the like. Some of thepresent polymers possess one or more property that make themparticularly suitable in the preparation of drug delivery devices. Suchproperties may include 1) the ability of the polymer to complex andprotect labile agents; 2) the ability to buffer the pH in the endosome;3) the ability to act as a “proton sponge” and cause endosomolysis; and4) the ability to neutralize the charge on negatively charged agents. Insome embodiments, the polymers are used to form microparticlescontaining the agent to be delivered. In some such embodiments, thediameter of the microparticles ranges from 500 nm to 50 micrometers,from 1 micrometer to 20 micrometers, or from 1 micrometer to 10micrometers. In other embodiments, the microparticles range from 1-5micrometers. The encapsulating polymer may be combined with otherpolymers (e.g., PEG, PLGA) to form the microspheres.

Methods of Preparing Microparticles

The microparticles may be prepared using various methods. Examples ofsuch methods include, but are not limited to, spray drying, single anddouble emulsion solvent evaporation, solvent extraction, phaseseparation, simple and complex coacervation, and other methods wellknown to those of ordinary skill in the art. In some embodiments, themethods for preparing the particles are the double emulsion process andspray drying methods. The conditions used in preparing themicroparticles may be altered to yield particles of a desired size orproperty (e.g., hydrophobicity, hydrophilicity, external morphology,“stickiness”, shape, etc.). The method of preparing the particle and theconditions (e.g., solvent, temperature, concentration, air flow rate,etc.) used may also depend on the agent being encapsulated and/or thecomposition of the polymer matrix.

Methods developed for making microparticles for delivery of encapsulatedagents are described in the literature (for example, please see Doubrow,M., Ed., “Microcapsules and Nanoparticles in Medicine and Pharmacy,” CRCPress, Boca Raton, 1992; Mathiowitz and Langer, J. Controlled Release5:13-22, 1987; Mathiowitz et al. Reactive Polymers 6:275-283, 1987;Mathiowitz et al. J. Appl. Polymer Sci. 35:755-774, 1988; each of whichis incorporated herein by reference).

If the particles prepared by any of the above methods have a size rangeoutside of the desired range, the particles can be sized, for example,using a sieve.

The present polymers and anions may also be combined together to formlayered interpolyelectrolyte complexes, similar to those disclosed inVazquez et al., J. Am. Chem. Soc. 124, 13992 (2002). Such layeredinterpolyelectrolyte complexes can also be produced by the methodsdisclosed in Vazquez et al., supra. The number of layers in such aninterpolyelectrolyte complex is not particularly limited. Additionally,different layers of these interpolyelectrolyte complexes can containdifferent polymers and/or anions. The present invention contemplatesthat these multilayer structures can be used for controlled release of adesired agent or delivery of multiple agents. As is understood by theskilled artisan, the film growth of the layered structure is primarilydictated by electrostatic interactions, hydrophobic interactions,hydrogen bonding, salt concentration, and solution pH. The presentlayered interpolyelectrolyte complexes can also be used to deliveranions to or transfect cells. In this embodiment the cells to betransfected can be placed or plated on the layered interpolyelectrolytecomplexes.

Agent

The agents to be delivered by the system of the present invention may betherapeutic, diagnostic, or prophylactic agents. Any anionic chemicalcompound to be administered to an individual may be delivered using theinterpolyelectrolyte complex. The agent may be a small molecule,organometallic compound, nucleic acid, protein, peptide, polynucleotide,metal, an isotopically labeled chemical compound, drug, vaccine,immunological agent, or the like.

In some embodiments, the agents are organic compounds withpharmaceutical activity. In another embodiment of the invention, theagent is a clinically used drug. In some such embodiments, the drug isan antibiotic, anti-viral agent, anesthetic, steroidal agent,anti-inflammatory agent, anti-neoplastic agent, antigen, vaccine,antibody, decongestant, antihypertensive, sedative, birth control agent,progestational agent, anti-cholinergic, analgesic, anti-depressant,anti-psychotic, β-adrenergic blocking agent, diuretic, cardiovascularactive agent, vasoactive agent, non-steroidal anti-inflammatory agent,nutritional agent, or the like, or combinations thereof.

In some embodiments of the present invention, the agent to be deliveredmay be a mixture of agents. For example, a local anesthetic may bedelivered in combination with an anti-inflammatory agent such as asteroid. Local anesthetics may also be administered with vasoactiveagents such as epinephrine. As a further example, an antibiotic may becombined with an inhibitor of the enzyme commonly produced by bacteriato inactivate an antibiotic (e.g., penicillin and clavulanic acid).

Diagnostic agents include gases; metals; commercially available imagingagents used in positron emissions tomography (PET), computer assistedtomography (CAT), single photon emission computerized tomography, x-ray,fluoroscopy, and magnetic resonance imaging (MRI); and contrast agents.Examples of suitable materials for use as contrast agents in MRI includegadolinium chelates, as well as iron, magnesium, manganese, copper, andchromium. Examples of materials useful for CAT and x-ray imaging includeiodine-based materials.

Prophylactic agents of the invention include, but are not limited to,antibiotics, nutritional supplements, and vaccines. Vaccines maycomprise isolated proteins or peptides, inactivated organisms andviruses, dead organisms and viruses, genetically altered organisms orviruses, and cell extracts. Prophylactic agents may be combined withinterleukins, interferon, cytokines, and adjuvants such as choleratoxin, alum, Freund's adjuvant, etc. Prophylactic agents includeantigens of such bacterial organisms as Streptococccus pneumoniae,Haemophilus influenzae, Staphylococcus aureus, Streptococcus pyrogenes,Corynebacterium diphtheriae, Listeria monocytogenes, Bacillus anthracis,Clostridium tetani, Clostridium botulinum, Clostridium perfringens,Neisseria meningitidis, Neisseria gonorrhoeae, Streptococcus mutans,Pseudomonas aeruginosa, Salmonella typhi, Haemophilus parainfluenzae,Bordetella pertussis, Francisella tularensis, Yersinia pestis, Vibriocholerae, Legionella pneumophila, Mycobacterium tuberculosis,Mycobacterium leprae, Treponema pallidum, Leptospirosis interrogans,Borrelia burgdorferi, Camphylobacter jejuni, and the like; antigens ofsuch viruses as smallpox, influenza A and B, respiratory syncytialvirus, parainfluenza, measles, HIV, varicella-zoster, herpes simplex 1and 2, cytomegalovirus, Epstein-Barr virus, rotavirus, rhinovirus,adenovirus, papillomavirus, poliovirus, mumps, rabies, rubella,coxsackieviruses, equine encephalitis, Japanese encephalitis, yellowfever, Rift Valley fever, hepatitis A, B, C, D, and E virus, and thelike; antigens of fungal, protozoan, and parasitic organisms such asCryptococcus neoformans, Histoplasma capsulatum, Candida albicans,Candida tropicalis, Nocardia asteroides, Rickettsia ricketsii,Rickettsia typhi, Mycoplasma pneumoniae, Chlamydial psittaci, Chlamydialtrachomatis, Plasmodium falciparum, Trypanosoma brucei, Entamoebahistolytica, Toxoplasma gondii, Trichomonas vaginalis, Schistosomamansoni, and the like. These antigens may be in the form of whole killedorganisms, peptides, proteins, glycoproteins, carbohydrates, orcombinations thereof.

Targeting Agents or Ligands

The micro- and nanoparticles of the invention may be modified to includetargeting agents since it is often desirable to target a particularcell, collection of cells, or tissue. A variety of targeting agents thatdirect pharmaceutical compositions to particular cells are known in theart (see, for example, Cotten et al. Methods Enzym. 217:618, 1993;incorporated herein by reference). The targeting agents may be includedthroughout the particle or may be only on the surface. The targetingagent may be a protein, peptide, carbohydrate, glycoprotein, lipid,small molecule, antibody, antibody fragment, receptor or the like. Thetargeting agent may be used to target specific cells or tissues or maybe used to promote endocytosis or phagocytosis of the particle. Examplesof targeting agents include, but are not limited to, antibodies,fragments of antibodies, low-density lipoproteins (LDLs), transferring,asialycoproteins, gp120 envelope protein of the human immunodeficiencyvirus (HIV), carbohydrates, receptor ligands, sialic acid, and the like.If the targeting agent is included throughout the particle, thetargeting agent may be included in the mixture that is used to form theparticles. If the targeting agent is only present on the surface, thetargeting agent may be associated with (i.e., by covalent, hydrophobic,hydrogen boding, van der Waals, or other interactions) the formedparticles using standard chemical techniques.

The amine groups on the branched PEI can also be conjugated eitherdirectly to the amine groups or via spacer molecules, with targetingligands and the like. Preferably, only a portion of the available aminegroups are coupled to the ligand or spacer/ligand such that the netcharge of the polymer is positive. The target ligands conjugated to thepolymer direct the polymer-nucleic acid/drug complex to bind to specifictarget cells and penetrate into such cells (tumor cells, liver cells,heamatopoietic cells, and the like). The target ligands can also be anintracellular targeting element, enabling the transfer of the nucleicacid/drug to be guided towards certain favored cellular compartments(mitochondria, nucleus, and the like). In certain embodiments, theligands can be sugar moieties coupled to the amino groups. Such sugarmoieties are preferably mono- or oligo-saccharides, such as galactose,glucose, fucose, fructose, lactose, sucrose, mannose, cellobiose,nytrose, triose, dextrose, trehalose, maltose, galactosamine,glucosamine, galacturonic acid, glucuronic acid, and gluconic acid.

The conjugation of an acid derivative of a sugar with the polymer ispreferred in some embodiments. In some such embodiments of the presentinvention, lactobionic acid (4-O-β-D-galactopyranosyl-D-gluconic acid)is coupled to the polymer. The galactosyl unit of lactose provides aconvenient targeting molecule for hepatocyte cells because of the highaffinity and avidity of the galactose receptor on these cells.

Other types of ligands that may be used include peptides such asantibodies or antibody fragments, cell receptors, growth factorreceptors, cytokine receptors, transferrin, epidermal growth factor(EGF), insulin, asialoorosomucoid, mannose-6-phosphate (monocytes),mannose (macrophage, some B cells), Lewis^(x) and sialyl Lewis^(x)(endothelial cells), N-acetyllactosamine (T cells), galactose (coloncarcinoma cells), and thrombomodulin (mouse lung endothelial cells),fusogenic agents such as polymixin B and hemaglutinin HA2,lysosomotrophic agents, nucleus localization signals (NLS) such asT-antigen, and the like.

Pharmaceutical Compositions

Once the microparticles or nanoparticles (interpolyelectrolyte complex,e.g. polymer complexed with anionic molecule or compound) have beenprepared, they may be combined with one or more pharmaceuticalexcipients to form a pharmaceutical composition that is suitable toadminister to animals. Animals include humans as well as non-humananimals, including, for example, mammals, birds, reptiles, amphibians,and fish. Preferably, the non-human animal is a mammal (e.g., a rodent,a mouse, a rat, a rabbit, a monkey, a dog, a cat, a primate, or a pig).An animal may be a transgenic animal. As would be appreciated by one ofskill in this art, the excipients may be chosen based on the route ofadministration as described below, the agent being delivered, timecourse of delivery of the agent, or other factors.

Pharmaceutical compositions of the present invention and for use inaccordance with the present invention may include a pharmaceuticallyacceptable excipient or carrier. As used herein, the term“pharmaceutically acceptable carrier” means a non-toxic, inert solid,semi-solid or liquid filler, diluent, encapsulating material orformulation auxiliary of any type. Some examples of materials which canserve as pharmaceutically acceptable carriers are sugars such aslactose, glucose, and sucrose; starches such as corn starch and potatostarch; cellulose and its derivatives such as sodium carboxymethylcellulose, ethyl cellulose, and cellulose acetate; powdered tragacanth;malt; gelatin; talc; excipients such as cocoa butter and suppositorywaxes; oils such as peanut oil, cottonseed oil; safflower oil; sesameoil; olive oil; corn oil and soybean oil; glycols such as propyleneglycol; esters such as ethyl oleate and ethyl laurate; agar; detergentssuch as Tween 80; buffering agents such as magnesium hydroxide andaluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline;Ringer's solution; ethyl alcohol; and phosphate buffer solutions, aswell as other non-toxic compatible lubricants such as sodium laurylsulfate and magnesium stearate, as well as coloring agents, releasingagents, coating agents, sweetening, flavoring and perfuming agents,preservatives and antioxidants can also be present in the composition,according to the judgment of the formulator. The pharmaceuticalcompositions of this invention can be administered to humans and/or toanimals, orally, rectally, parenterally, intracisternally,intravaginally, intranasally, intraperitoneally, topically (as bypowders, creams, ointments, or drops), bucally, or as an oral or nasalspray.

Liquid dosage forms for oral administration include pharmaceuticallyacceptable emulsions, microemulsions, solutions, suspensions, syrups,and elixirs. In addition to the active ingredients (i.e.,microparticles, nanoparticles, polynucleotide/polymer complexes), theliquid dosage forms may contain inert diluents commonly used in the artsuch as, for example, water or other solvents, solubilizing agents andemulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate,ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol,1,3-butylene glycol, dimethylformamide, oils (in particular, cottonseed,groundnut, corn, germ, olive, castor, and sesame oils), glycerol,tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid estersof sorbitan, and mixtures thereof. Besides inert diluents, the oralcompositions can also include adjuvants such as wetting agents,emulsifying and suspending agents, sweetening, flavoring, and perfumingagents.

Injectable preparations, for example, sterile injectable aqueous oroleaginous suspensions may be formulated according to the known artusing suitable dispersing or wetting agents and suspending agents. Thesterile injectable preparation may also be a sterile injectablesolution, suspension, or emulsion in a nontoxic parenterally acceptablediluent or solvent, for example, as a solution in 1,3-butanediol. Amongthe acceptable vehicles and solvents that may be employed are water,Ringer's solution, U.S.P. and isotonic sodium chloride solution. Inaddition, sterile, fixed oils are conventionally employed as a solventor suspending medium. For this purpose any bland fixed oil may beemployed including synthetic mono- or diglycerides. In addition, fattyacids such as oleic acid may be used in the preparation of injectables.In some embodiments, the particles are suspended in a carrier fluidcomprising 1% (w/v) sodium carboxymethyl cellulose and 0.1% (v/v) Tween80.

The injectable formulations can be sterilized, for example, byfiltration through a bacteria-retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved or dispersed in sterile water or other sterile injectablemedium prior to use.

Compositions for rectal or vaginal administration are preferablysuppositories which can be prepared by mixing the particles withsuitable non-irritating excipients or carriers such as cocoa butter,polyethylene glycol, or a suppository wax which are solid at ambienttemperature but liquid at body temperature and therefore melt in therectum or vaginal cavity and release the microparticles.

Solid dosage forms for oral administration include capsules, tablets,pills, powders, and granules. In such solid dosage forms, the particlesare mixed with at least one inert, pharmaceutically acceptable excipientor carrier such as sodium citrate or dicalcium phosphate and/or a)fillers or extenders such as starches, lactose, sucrose, glucose,mannitol, and silicic acid, b) binders such as, for example,carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone,sucrose, and acacia, c) humectants such as glycerol, d) disintegratingagents such as agar-agar, calcium carbonate, potato or tapioca starch,alginic acid, certain silicates, and sodium carbonate, e) solutionretarding agents such as paraffin, f) absorption accelerators such asquaternary ammonium compounds, g) wetting agents such as, for example,cetyl alcohol and glycerol monostearate, h) absorbents such as kaolinand bentonite clay, and i) lubricants such as talc, calcium stearate,magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate,and mixtures thereof. In the case of capsules, tablets, and pills, thedosage form may also comprise buffering agents.

Solid compositions of a similar type may also be employed as fillers insoft and hard-filled gelatin capsules using such excipients as lactoseor milk sugar as well as high molecular weight polyethylene glycols andthe like.

The solid dosage forms of tablets, dragees, capsules, pills, andgranules can be prepared with coatings and shells such as entericcoatings and other coatings well known in the pharmaceutical formulatingart. They may optionally contain opacifying agents and can also be of acomposition that they release the active ingredient(s) only, orpreferentially, in a certain part of the intestinal tract, optionally,in a delayed manner. Examples of embedding compositions which can beused include polymeric substances and waxes.

Solid compositions of a similar type may also be employed as fillers insoft and hard-filled gelatin capsules using such excipients as lactoseor milk sugar as well as high molecular weight polyethylene glycols andthe like.

Dosage forms for topical or transdermal administration of anpharmaceutical composition include ointments, pastes, creams, lotions,gels, powders, solutions, sprays, inhalants, or patches. The particlesare admixed under sterile conditions with a pharmaceutically acceptablecarrier and any needed preservatives or buffers as may be required.Ophthalmic formulation, ear drops, and eye drops are also contemplatedas being within the scope of this invention.

The ointments, pastes, creams, and gels may contain, in addition to theparticles of this invention, excipients such as animal and vegetablefats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives,polyethylene glycols, silicones, bentonites, silicic acid, talc, andzinc oxide, or mixtures thereof.

Powders and sprays can contain, in addition to the particles of thisinvention, excipients such as lactose, talc, silicic acid, aluminumhydroxide, calcium silicates, and polyamide powder, or mixtures of thesesubstances. Sprays can additionally contain customary propellants suchas chlorofluorohydrocarbons.

Transdermal patches have the added advantage of providing controlleddelivery of a compound to the body. Such dosage forms can be made bydissolving or dispensing the microparticles or nanoparticles in a propermedium. Absorption enhancers can also be used to increase the flux ofthe compound across the skin. The rate can be controlled by eitherproviding a rate controlling membrane or by dispersing the particles ina polymer matrix or gel.

The present invention also provides methods of administering the presentpolymers and complexes. Generally, these methods can involve contactingan interpolyelectrolyte complex of the present invention with one ormore cells, such as those that make up a tissue. In one embodiment, theinterpolyelectrolyte complex is administered to an animal. Theinterpolyelectrolyte complex can be administered in any suitable manner,such as in the manner and formulations described above. In someembodiments, in solution the side chain esters slowly hydrolyze,resulting in the release of the anionic component that is boundelectrostatically to the polymer. Some of the present methods can beused to transfect cells.

The present invention also provides methods for delivering an anioniccompound to a cell or tissue. The present methods involve contacting acomposition that includes a present interpolyelectrolyte complex with atarget cell thereby allowing the target cell to uptake the composition.The polymer of the present invention is designed such that when theinterpolyelectrolyte complex enters the target cell, one or more of theremovable functional group(s) is removed from the dynamic charge statecationic polymer which decreases the cationic charge density of thedynamic charge state cationic polymer. The decrease in the cationiccharge density of the polymer is caused by the introduction of anioniccharges which promotes dissociation of the interpolyelectrolyte complexinto the dynamic charge state cationic polymer and the anionic moleculeallowing for delivery of the anionic molecule to the target cell or cellcompartment, such as an endosome, cytosol or nucleus of the cell. Insome methods, at least one of the one or more of the removablefunctional group(s) is removed from the dynamic charge state cationicpolymer in a nucleus, endosome or cytosol of the target cell. In thismanner, the interpolyelectrolyte complex can dissociate primarily in thedesired compartment of the target cell and deliver the anionic moleculeto the target cell compartment. The present methods can also involveproviding the interpolyelectrolyte complex and/or preparing theinterpolyelectrolyte complex. Generally, the interpolyelectrolytecomplex will be prepared by mixing the dynamic charge state cationicpolymer with the anionic molecule thereby allowing formation of theinterpolyelectrolyte complex. In the methods where the anionic moleculeis DNA, the DNA can be delivered to the nucleus of the cell so that itis stably incorporated into the genome of the target cell. In otherembodiments, the DNA is not stably incorporated into the genome of thetarget cell.

In the present methods, the target cell or tissue can be in vitro or invivo. Where the target cell or tissue is in vivo, theinterpolyelectrolyte complex can be administered to a mammal. In someembodiments of the present methods, the cell is a eukaryotic cell.

In the present methods and polymers, removal of the one or more of theremovable functional group(s) from the dynamic charge state cationicpolymer can be at least partially hydrolytic, partially enzymatic and/orpartially photolytic removal. The present polymers and methods can alsobe designed so that removal of the one or more of the removablefunctional groups from the dynamic charge state cationic polymer occursat a substantially constant rate or does not occur at a constant rate.Accordingly, in the present methods, the majority, or substantially all,of the anions can be delivered to the desired part of the cell, such asthe nucleus, endosome or cytosol.

The present invention also provides kits for carrying out the methodsdescribed herein. In one embodiment, the kit is made up of instructionsfor carrying out any of the methods described herein. The instructionscan be provided in any intelligible form through a tangible medium, suchas printed on paper, computer readable media, or the like. The presentkits can also include one or more reagents, buffers, media, nucleicacids, agents and/or disposable equipment in order to readily facilitateimplementation of the present methods. Examples of kit components can befound in the description above and in the following examples. Such kitsmay be used in hospitals, clinics, physician's offices or in patients'homes to facilitate the co-administration of the enhancing and targetagents. The kits may also include as an insert printed dosinginformation for the co-administration of the enhancing and targetagents.

These and other aspects of the present invention will be furtherappreciated upon consideration of the following Examples, which areintended to illustrate certain particular embodiments of the inventionbut are not intended to limit its scope, as defined by the claims.

Definitions

The following are terms used in the present application:

The term “alkyl” as used herein refers to saturated, straight- orbranched-chain hydrocarbon radicals derived from a hydrocarbon moietycontaining between one and twenty carbon atoms by removal of a singlehydrogen atom. In some embodiments, alkyl groups have from 1 to 12 orfrom 1 to 8 carbon atoms. Examples of alkyl radicals include, but arenot limited to, methyl, ethyl, propyl, isopropyl, n-butyl, tert-butyl,n-pentyl, neopentyl, n-hexyl, n-heptyl, n-octyl, n-decyl, n-undecyl, anddodecyl. A “cycloalkyl” group is a cyclic alkyl group typicallycontaining from 3 to 8 ring members such as, but not limited to, acyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, orcyclooctyl group.

The term “alkoxy” as used herein refers to an alkyl group, as previouslydefined, attached to the parent molecular moiety through an oxygen atom.Examples include, but are not limited to, methoxy, ethoxy, propoxy,isopropoxy, n-butoxy, tert-butoxy, neopentoxy, and n-hexoxy groups.

The term “alkenyl” denotes a monovalent group derived from a hydrocarbonmoiety having at least one carbon-carbon double bond by the removal of asingle hydrogen atom. Alkenyl groups include, for example, ethenyl,propenyl, butenyl, 1-methyl-2-buten-1-yl, and the like.

The term “alkynyl” as used herein refers to a monovalent group derivedform a hydrocarbon having at least one carbon-carbon triple bond by theremoval of a single hydrogen atom. Representative alkynyl groups includeethynyl, 2-propynyl (propargyl), 1-propynyl, and the like.

The terms “alkylamino”, “dialkylamino”, and “trialkylamino” as usedherein refer to amino groups respectively having one, two, or threealkyl groups, as previously defined, attached to the parent molecularmoiety through a nitrogen atom. The term “alkylamino” refers to a grouphaving the structure —NHR′ wherein R′ is an alkyl group, as previouslydefined. The term “dialkylamino” refers to a group having the structure—NR′R″, where R′ and R″ are each independently selected from the groupconsisting of alkyl groups. Finally, the term “trialkylamino” refers toa group having the structure —NR′R″R′″, where R′, R″, and R′″ are eachindependently selected from alkyl groups. Additionally, R′, R″, and/orR′″, taken together, may optionally be a —(CH₂)_(k)— group where k is aninteger ranging from 2 to 6. Examples of “alkylamino”, “dialkylamino”,and “trialkylamino” groups include, but are not limited to, methylamino,dimethylamino, ethylamino, diethylamino, diethylaminocarbonyl,methylethylamino, isopropylamino, piperidino, trimethylamino, andpropylamino groups.

The terms “alkylthioether” and “thioalkoxy” refer to an alkyl group, aspreviously defined, attached to the parent molecular moiety through asulfur atom.

The term “aryl” as used herein refers to carbocyclic ring systems havingat least one aromatic ring including, but not limited to, phenyl,naphthyl, tetrahydronaphthyl, indanyl, and indenyl groups, and the like.Aryl groups can be unsubstituted or substituted with substituentsselected from the group consisting of branched and unbranched alkyl,alkenyl, alkynyl, haloalkyl, alkoxy, thioalkoxy, amino, alkylamino,dialkylamino, trialkylamino, acylamino, cyano, hydroxy, halo, mercapto,nitro, carboxyaldehyde, carboxy, alkoxycarbonyl, and carboxamide. Inaddition, substituted aryl groups include tetrafluorophenyl andpentafluorophenyl.

The terms “heterocyclic” and “heterocyclyl”, as used herein, refer to anon-aromatic partially unsaturated or fully saturated 3- to 10-memberedring system, which includes single rings of 3 to 8 atoms in size and bi-and tri-cyclic ring systems which may include aromatic six-membered arylor aromatic heterocyclic groups fused to a non-aromatic ring. Theseheterocyclic and heterocyclyl rings and groups include those having fromone to three heteroatoms independently selected from oxygen, sulfur, andnitrogen, in which the nitrogen and sulfur heteroatoms may optionally beoxidized and the nitrogen heteroatom may optionally be quaternary.

The terms “aromatic heterocyclic” and “aromatic heterocyclyl”, as usedherein, refer to a cyclic aromatic radical having from five to ten ringatoms of which one ring atom is selected from sulfur, oxygen, andnitrogen; zero, one, or two ring atoms are additional heteroatomsindependently selected from sulfur, oxygen, and nitrogen; and theremaining ring atoms are carbon, the radical being joined to the rest ofthe molecule via any of the ring atoms. Examples of such aromaticheterocyclyl groups include, but are not limited to, pyridyl, pyrazinyl,pyrimidinyl, pyrrolyl, pyrazolyl, imidazolyl, thiazolyl, oxazolyl,isooxazolyl, thiadiazolyl, oxadiazolyl, thiophenyl, furanyl, quinolinyl,and isoquinolinyl groups, and the like.

Specific heterocyclic and aromatic heterocyclic groups that may beincluded in the compounds of the invention include:3-methyl-4-(3-methylphenyl)piperazine, 3 methylpiperidine,4-(bis-(4-fluorophenyl)methyl)piperazine, 4-(diphenylmethyl)piperazine,4-(ethoxycarbonyl)piperazine, 4-(ethoxycarbonylmethyl)piperazine,4-(phenylmethyl)piperazine, 4-(1-phenylethyl)piperazine,4-(1,1-dimethylethoxycarbonyl)piperazine,4-(2-(bis-(2-propenyl)amino)ethyl)piperazine,4-(2-(diethylamino)ethyl)piperazine, 4-(2-chlorophenyl)piperazine,4-(2-cyanophenyl)piperazine, 4-(2-ethoxyphenyl)piperazine,4-(2-ethylphenyl)piperazine, 4-(2-fluorophenyl)piperazine,4-(2-hydroxyethyl)piperazine, 4-(2-methoxyethyl)piperazine,4-(2-methoxyphenyl)piperazine, 4-(2-methylphenyl)piperazine,4-(2-methylthiophenyl)piperazine, 4-(2-nitrophenyl)piperazine,4-(2-nitrophenyl)piperazine, 4-(2-phenylethyl)piperazine,4-(2-pyridyl)piperazine, 4-(2-pyrimidinyl)piperazine,4-(2,3-dimethylphenyl)piperazine, 4-(2,4-difluorophenyl)piperazine,4-(2,4-dimethoxyphenyl)piperazine, 4-(2,4-dimethylphenyl)piperazine,4-(2,5-dimethylphenyl)piperazine, 4-(2,6-dimethylphenyl)piperazine,4-(3-chlorophenyl)piperazine, 4-(3-methylphenyl)piperazine,4-(3-trifluoromethylphenyl)piperazine, 4-(3,4-dichlorophenyl)piperazine,4-3,4-dimethoxyphenyl)piperazine, 4-(3,4-dimethylphenyl)piperazine,4-(3,4-methylenedioxyphenyl)piperazine,4-(3,4,5-trimethoxyphenyl)piperazine, 4-(3,5-dichlorophenyl)piperazine,dimethoxyphenyl)piperazine, 4-(4-(phenylmethoxy)phenyl)piperazine,4-(4-(3,1-dimethylethyl)phenylmethyl)piperazine,4-(4-chloro-3-trifluoromethylphenyl)piperazine,4-(4-chlorophenyl)-3-methylpiperazine, 4-(4-chlorophenyl)piperazine,4-(4-chlorophenyl)piperazine, 4-(4-chlorophenylmethyl)piperazine,4-(4-fluorophenyl)piperazine, 4-(4-methoxyphenyl)piperazine,4-(4-methylphenyl)piperazine, 4-(4-nitrophenyl)piperazine,4-(4-trifluoromethylphenyl)piperazine, 4-cyclohexylpiperazine,4-ethylpiperazine, 4-hydroxy-4-(4-chlorophenyl)methylpiperidine,4-hydroxy-4-phenylpiperidine, 4-hydroxypyrrolidine, 4-methylpiperazine,4-phenylpiperazine, 4-piperidinylpiperazine,4-(2-furanyl)carbonyl)piperazine,4-((1,3-dioxolan-5-yl)methyl)piperazine-,6-fluoro-1,2,3,4-tetrahydro-2-methylquinoline, 1,4-diazacylcloheptane,2,3-dihydroindolyl, 3,3-dimethylpiperidine, 4,4-ethylenedioxypiperidine,1,2,3,4-tetrahydroisoquinoline, 1,2,3,4-tetrahydroquinoline,azacyclooctane, decahydroquinoline, piperazine, piperidine, pyrrolidine,thiomorpholine, and triazole.

The term “hydrocarbon”, as used herein, refers to any chemical groupcomprising hydrogen and carbon. The hydrocarbon may be substituted orunsubstituted. The hydrocarbon may be unsaturated, saturated, branched,unbranched, cyclic, polycyclic, or heterocyclic. Illustrativehydrocarbons include, for example, methyl, ethyl, n-propyl, iso-propyl,cyclopropyl, allyl, vinyl, n-butyl, tert-butyl, ethynyl, cyclohexyl,methoxy, diethylamino, and the like. As would be known to one skilled inthis art, all valencies must be satisfied in making any substitutions.

The terms “substituted”, whether preceded by the term “optionally” ornot, and “substituent”, as used herein, refer to the ability, asappreciated by one skilled in this art, to change one functional groupfor another functional group provided that the valency of all atoms ismaintained. When more than one position in any given structure may besubstituted with more than one substituent selected from a specifiedgroup, the substituent may be either the same or different at everyposition. The substituents may also be further substituted (e.g., anaryl group substituent may be further substituted. For example, a nonlimiting example is an aryl group that may be further substituted with,for example, a fluorine group at one or more position.

When two entities are “associated with” one another as described herein,they are linked by a direct or indirect covalent or non-covalentinteraction. Preferably, the association is covalent. Desirablenon-covalent interactions include hydrogen bonding, van der Waalsinteractions, hydrophobic interactions, magnetic interactions,electrostatic interactions, etc.

As used herein, “biodegradable” compounds are those that, whenintroduced into cells, are broken down by the cellular machinery or byhydrolysis into components that the cells can either reuse or disposeof, in some cases without significant toxic effect on the cells (e.g.,fewer than about 20% of the cells are killed when the components areadded to cells in vitro). The components preferably do not induceinflammation or other adverse effects in vivo. In certain embodiments,the chemical reactions relied upon to break down the biodegradablecompounds are uncatalyzed.

A “labile bond” is a covalent bond that is capable of being selectivelybroken. That is, a labile bond may be broken in the presence of othercovalent bonds without the breakage of other covalent bonds. Forexample, a disulfide bond is capable of being broken in the presence ofthiols without cleavage of any other bonds, such as carbon-carbon,carbon-oxygen, carbon-sulfur, carbon-nitrogen bonds, which may also bepresent in the molecule. “Labile” also means cleavable.

A “labile linkage” is a chemical compound that contains a labile bondand provides a link or spacer between two other groups. The groups thatare linked may be chosen from compounds such as biologically activecompounds, membrane active compounds, compounds that inhibit membraneactivity, functional reactive groups, monomers, and cell targetingsignals. The spacer group may contain chemical moieties chosen from agroup that includes alkanes, alkenes, esters, ethers, glycerol, amide,saccharides, polysaccharides, and heteroatoms such as oxygen, sulfur, ornitrogen. The spacer may be electronically neutral, may bear a positiveor negative charge, or may bear both positive and negative charges withan overall charge of neutral, positive or negative.

In general, the “effective amount” of an active agent or drug deliverydevice refers to the amount necessary to elicit the desired biologicalresponse. As will be appreciated by those of ordinary skill in this art,the effective amount of an agent or device may vary depending on suchfactors as the desired biological endpoint, the agent to be delivered,the composition of the encapsulating matrix, the target tissue, etc. Forexample, the effective amount of microparticles containing an antigen tobe delivered to immunize an individual is the amount that results in animmune response sufficient to prevent infection with an organism havingthe administered antigen.

As used herein, “peptide”, means peptides of any length and includesproteins. The terms “polypeptide” and “oligopeptide” are used hereinwithout any particular intended size limitation, unless a particularsize is otherwise stated. The only limitation to the peptide or proteindrug which may be utilized is one of functionality. The terms “protein”and “peptide” may be used interchangeably. Peptide may refer to anindividual peptide or a collection of peptides. Peptides preferablycontain only natural amino acids, although non-natural amino acids(i.e., compounds that do not occur in nature but that can beincorporated into a polypeptide chain; see, for example,http://www.cco.caltech.edu/.about.da-dgrp/Unnatstruct.gif, whichdisplays structures of non-natural amino acids that have beensuccessfully incorporated into functional ion channels) and/or aminoacid analogs as are known in the art may alternatively be employed.Also, one or more of the amino acids in an peptide may be modified, forexample, by the addition of a chemical entity such as a carbohydrategroup, a phosphate group, a farnesyl group, an isofarnesyl group, afatty acid group, a linker for conjugation, functionalization, or othermodification, etc. In some embodiments, the modifications of the peptidelead to a more stable peptide (e.g., greater half-life in vivo). Thesemodifications may include cyclization of the peptide, the incorporationof D-amino acids, etc. Typical of peptides that can be utilized arethose selected from the group consisting of oxytocin, vasopressin,adrenocorticotrophic hormone, epidermal growth factor, prolactin,luliberin or luteinising hormone releasing hormone, growth hormone,growth hormone releasing factor, insulin, somatostatin, glucagon,interferon, gastrin, tetragastrin, pentagastrin, urogastroine, secretin,calcitonin, enkephalins, endorphins, angiotensins, renin, bradykinin,bacitracins, polymixins, colistins, tyrocidin, grarnicidines, andsynthetic analogues, modifications and pharmacologically activefragments thereof, monoclonal antibodies and soluble vaccines.

The terms “polynucleotide” and “oligonucleotide” refer to a polymer ofnucleotides. Typically, a polynucleotide comprises at least threenucleotides. The polymer may include natural nucleosides (i.e.,adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine,deoxythymidine, deoxyguanosine, and deoxycytidine), nucleoside analogs(e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine,3-methyl adenosine, C⁵-propynylcytidine, C⁵-propynyluridine,C⁵-bromouridine, C⁵-fluorouridine, C⁵-iodouridine, C⁵-methylcytidine,7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine,O(6)-methylguanine, and 2-thiocytidine), chemically modified bases,biologically modified bases (e.g., methylated bases), intercalatedbases, modified sugars (e.g., 2′-fluororibose, ribose, 2′-deoxyribose,arabinose, and hexose), or modified phosphate groups (e.g.,phosphorothioates and 5′-N-phosphoramidite linkages).

As used herein, the term “small molecule” refers to organic compounds,whether naturally-occurring or artificially created (e.g., via chemicalsynthesis) that have relatively low molecular weight and that are notproteins, polypeptides, or nucleic acids. Typically, small moleculeshave a molecular weight of less than about 1500 g/mol. Also, smallmolecules typically have multiple carbon-carbon bonds. Knownnaturally-occurring small molecules include, but are not limited to,penicillin, erythromycin, taxol, cyclosporin, and rapamycin. Knownsynthetic small molecules include, but are not limited to, ampicillin,methicillin, sulfamethoxazole, and sulfonamides.

As used herein, “administering”, and similar terms means delivering thecomposition to the individual being treated. In some instances thecomposition is capable of being circulated systemically where thecomposition binds to a target cell and is taken up by endocytosis. Thus,the composition is preferably administered to the individualsystemically, typically by subcutaneous, intramuscular, intravenous, orintraperitoneal administration. Injectables for such use can be preparedin conventional forms, either as a liquid solution or suspension, or ina solid form that is suitable for preparation as a solution orsuspension in a liquid prior to injection, or as an emulsion. Suitableexcipients include, for example, water, saline, dextrose, glycerol,ethanol, and the like; and if desired, minor amounts of auxiliarysubstances such as wetting or emulsifying agents, buffers, and the likecan be added.

A “pharmaceutically acceptable salt” includes a salt with an inorganicbase, organic base, inorganic acid, organic acid, or basic or acidicamino acid. As salts of inorganic bases, the invention includes, forexample, alkali metals such as sodium or potassium; alkaline earthmetals such as calcium and magnesium or aluminum; and ammonia. As saltsof organic bases, the invention includes, for example, trimethylamine,triethylamine, pyridine, picoline, ethanolamine, diethanolamine, andtriethanolamine. As salts of inorganic acids, the instant inventionincludes, for example, hydrochloric acid, hydroboric acid, nitric acid,sulfuric acid, and phosphoric acid. As salts of organic acids, theinstant invention includes, for example, formic acid, acetic acid,trifluoroacetic acid, fumaric acid, oxalic acid, lactic acid, tartaricacid, maleic acid, citric acid, succinic acid, malic acid,methanesulfonic acid, benzenesulfonic acid, and p-toluenesulfonic acid.As salts of basic amino acids, the instant invention includes, forexample, arginine, lysine and ornithine. Acidic amino acids include, forexample, aspartic acid and glutamic acid.

EXAMPLES Polymer Synthesis

The conjugate addition of methyl acrylate to linear polyethylene iminewas performed in the following general manner: To a solution of linearpolyethylene imine (5 wt % in methanol) was added methyl acrylate(Aldrich). Polyethylene imine was obtained by hydrolyzing polyethyloxazoline (Polysciences, Warrington, Pa.). Typically, the amount ofmethyl acrylate added was varied (e.g., from 0.25 to 1.2 equivalentsrelative to amine functionality in the PEI) to achieve desired molepercent substitutions (Table 1). The reaction mixtures were heated to40° C. and stirred overnight. The resulting products were concentratedby rotary evaporation, dissolved in dichloromethane, and precipitated byhexanes. The precipitate was dried under vacuum to yield the desiredproduct in near quantitative yield. Purified polymers were characterizedby nuclear magnetic resonance spectroscopy (NMR), gel permeationchromatography (GPC), and elemental analysis to determine percentfunctionalization. As a representative example for a polymer with >90%substitution: ¹H NMR (CDCl₃): δ (ppm)=2.45 (t, —CH₂CH₂N(CH₂CH₂CO₂CH₃));2.5 (s, —CH₂CH₂N(CH₂CH₂CO₂CH₃)); 2.8 (t, —CH₂CH₂N(CH₂CH₂CO₂CH₃)); 3.7(s, —CH₂CH₂N(CH₂CH₂CO₂CH₃)). The molecular weights of three sets offunctionalized polymers prepared from three different samples of PEIwere 8,200 g/mol, 14,000 g/mol, or 34,000 g/mol, as determined by GPC.

Equivalents of Methyl Acrylate^(a) Percent Polymer Substitution^(b) 0.25eq 24.8% 0.50 eq 45.8% 0.75 eq 79.8%  1.2 eq 90.9% ^(a)Relative to aminegroups in PEI. ^(b)Determined by elemental analysis.

In the above table, the percent substitution as determined by elementalanalysis is shown for the 14,000 M_(w) polymer.

Formation of DNA/Polymer Complexes and Agarose Gel Retardation Assays

DNA/polymer complexes were formed by adding 50 μL of a plasmid DNAsolution (2 μg/50 μl in water) to a gently vortexing solution of polymer(50 μL in 20 mM HEPES, pH=7.2). In each case, the concentration ofpolymer in this volume of buffer was adjusted to yield a desiredDNA/polymer weight ratio (e.g., 1:1, 1:2, 1:3, etc). These samples wereincubated at room temperature for 30 minutes, after which 20 μL of eachsample was mixed with a loading buffer and analyzed on a 1% agarose gel(HEPES, 20 mM, pH=7.2, 65V, 30 min). DNA bands were visualized byethidium bromide staining. Samples used to evaluate time courses ofDNA/polymer interaction were prepared as described above and split into4 equivalent 25 μL samples. These samples were incubated at 37° C. for0, 24, 48, and 72 hours and analyzed by agarose gel electrophoresis asdescribed above.

The results of these gel retardation assays are shown in FIGS. 1-11.These figures show the electrophoretic mobility of DNA in the presenceof particular polymers of different molecular weights and percentfunctionalization. As an example, FIG. 1 shows the migration of DNAcomplexed to PEI 91% substituted with methyl acrylate (M_(w)=14,000g/mol; polydispersity index (PDI)=2.25 by GPC) as a function of time.The PDI is a measure of how broad the molecular weight distribution isfor a given polymer sample. After initial complexation, DNA is bound bypolymer and retained in the wells at all weight ratios higher than 1:1DNA/polymer. At 24 hours, DNA is partially released and at 48 hours, DNAis completely released at all DNA/polymer ratios, as compared to acontrol experiment employing only DNA. Subsequent Figures show analogousrelease experiments using polymers of different molecular weights orpercent functionalization. It can be seen from the FIGS. that increasingthe percent substitution of the polymer results in earlier and morequantitative release of DNA over the range of DNA/polymer ratios andtimes employed. FIGS. 1-10 demonstrate that varying the molecularweights of these charge dynamic polymers also has a significantinfluence on the kinetics of DNA release. In these examples, lowermolecular weight charge dynamic polymers release DNA more rapidly thanhigher molecular weight polymers at analogous percent substitutions. NMRsuggests that the percent substitution of the polymer used in FIG. 10was about 50% and not the target 25%. FIG. 11 demonstrates theinteraction of completely hydrolyzed samples of the charge dynamicpolymers described above with DNA.

Dynamic Light Scattering

DNA/polymer complexes were formed as described above for agarose gelretardation assays. Samples were diluted with 900 μL of HEPES (20 mM,pH=7.2, total volume=1 mL). Average effective diameters were determinedat 25° C. Correlation functions were collected at a scattering angle of90°, and particle sizes were calculated using the viscosity andrefractive index of pure water at 25° C. Particle sizes were calculatedand expressed as effective diameters assuming a lognormal distribution.

General Protocol for Cell Transfection Assays

Transfection assays were performed in triplicate in the followinggeneral manner in 96-, 24-, or 6-well cell culture plates. Cells weregrown in 96-well plates at initial seeding densities of 15,000cells/well in 200 pt of growth medium (90% Dulbecco's modified Eagle'smedium, 10% fetal bovine serum, penicillin 100 units/mL, streptomycin100 μg/mL). Cells were grown for 24 hours in an incubator, after whichthe growth medium was removed and replaced with 200 μL of serum-free orserum-containing medium. DNA/polymer complexes were prepared asdescribed above for gel electrophoresis assays (e.g., using a plasmidcontaining the firefly luciferase reporter gene (pCMV-Luc)) over therange of different DNA/polymer complexes to be evaluated. An appropriatevolume of each sample was added to the cells using a pipette. Thisvolume was typically varied to provide desired concentrations of DNA orDNA/polymer complexes in each well. Controls employing poly(ethyleneimine) (PEI) or commercially-available lipid transfection reagents wereprepared in a similar manner and included along with DNA and no-DNAcontrols. Cells were incubated for 4 hours, after which the growthmedium was removed and replaced with 200 μL of growth medium. Cells wereincubated for an additional period of time (e.g., varied between 24 to72 hours) and luciferase expression was determined using a commerciallyavailable assay kit. Luminescence was quantified in white, solid-bottompolypropylene 96-well plates using a 96-well bioluminescence platereader. Luminescence was expressed in relative light units, totalprotein, or light units normalized to total cell protein.

Cytotoxicity Assays

Cytotoxicity assays were performed in 96-well plates according to thefollowing general protocol: Cells were grown in 96-well plates atinitial seeding densities of 10,000 cells/well in 200 μL growth medium(90% Dulbecco's modified Eagle's medium, 10% fetal bovine serum,penicillin 100 units/mL, streptomycin 100 μg/mL). Cells were grown for24 hours, after which the growth medium was removed and replaced with180 μL of serum-free medium. Desired amounts of polymer were added in 20μL aliquots. Samples were incubated at 37° C. for 5 hours, and themetabolic activity of each well was determined using a MTT/thiazolylblue assay. Generally: to each well was added 25 μL of a 5 mg/mLsolution of MTT stock solution in sterile PBS buffer. The samples wereincubated at 37° C. for 2 hours, and 100 μL of extraction buffer (20%w/v SDS in DMF/water (1:1), pH=4.7) was added to each well. Samples wereincubated at 37° C. for 24 hours. Optical absorbance was measured at 560nm with a microplate reader and expressed as a percent relative tocontrol cells. This assay was also performed by treating cells withDNA/polymer complexes rather than just dissolved polymer.

The present methods may be carried out by performing any of the stepsdescribed herein, either alone or in various combinations. The presentcompounds may also have any or all of the components described herein.One skilled in the art will recognize that all embodiments of thepresent invention are capable of use with all other embodiments of theinvention described herein. Additionally, one skilled in the art willrealize that the present invention also encompasses variations of thepresent methods and compositions that specifically exclude one or moreof the steps, components or groups described herein.

As will be understood by one skilled in the art, for any and allpurposes, particularly in terms of providing a written description, allranges disclosed herein also encompass any and all possible subrangesand combinations of subranges thereof. Any listed range can be easilyrecognized as sufficiently describing and enabling the same range beingbroken down into at least equal halves, thirds, quarters, fifths,tenths, etc. As a non-limiting example, each range discussed herein canbe readily broken down into a lower third, middle third and upper third,etc. As will also be understood by one skilled in the art all languagesuch as “up to,” “at least,” “greater than,” “less than,” “more than”and the like include the number recited and refer to ranges which can besubsequently broken down into subranges as discussed above. In the samemanner, all ratios disclosed herein also include all subratios fallingwithin the broader ratio.

One skilled in the art will also readily recognize that where membersare grouped together in a common manner, such as in a Markush group, thepresent invention encompasses not only the entire group listed as awhole, but each member of the group individually and all possiblesubgroups of the main group. Accordingly, for all purposes, the presentinvention encompasses not only the main group, but also the main groupabsent one or more of the group members. The present invention alsoenvisages the explicit exclusion of one or more of any of the groupmembers in the claimed invention.

All references, patents and publications disclosed herein arespecifically incorporated by reference herein in their entireties andfor all purposes as if fully set forth herein.

Unless otherwise specified, “a” or “an” means “one or more”.

While certain specific embodiments have been illustrated and described,it should be understood that changes and modifications can be madetherein in accordance with ordinary skill in the art without departingfrom the invention in its broader aspects as defined in the followingclaims.

1-57. (canceled)
 58. A multilayered interpolyelectrolyte complexcomprising a first layer comprising a first interpolyelectrolyte complexand a second layer comprising a second interpolyelectrolyte complex,wherein each of the first and second interpolyelectrolyte complexes,independently from one another, comprise: a. a dynamic charge statecationic polymer having a polymeric backbone formed from monomericunits, and one or more removable functional groups attached to thepolymeric backbone through one or more labile linkages, wherein the oneor more removable functional groups are selected from the groupconsisting of an alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclic,aryl, and heteroaryl group; and b. one or more anions complexed to saidcationic polymer; wherein the overall positive charge of the cationicpolymer decreases when one or more of the removable functional groups isremoved from the polymeric backbone, thereby causing said one or moreanions to disassociate from the cationic polymer, wherein the cationicpolymer and one or more anions in the first interpolyelectrolyte complexcan be the same or different from the cationic polymer and one or moreanions in the second interpolyelectrolyte complex.
 59. The multilayeredinterpolyelectrolyte complex of claim 58 wherein the cationic polymerand one or more anions in the first interpolyelectrolyte complex are thesame as the cationic polymer and one or more anions in the secondinterpolyelectrolyte complex.
 60. The multilayered interpolyelectrolytecomplex of claim 58 wherein the cationic polymer and one or more anionsin the first interpolyelectrolyte complex are different from thecationic polymer and one or more anions in the secondinterpolyelectrolyte complex.
 61. The multilayered interpolyelectrolytecomplex of claim 58 wherein the polymeric backbone in the first andsecond interpolyelectrolyte complexes, independently from one another,are selected from the group consisting of polyethylene imine,polylysine, polyornithine, poly(propylene imine), poly(allyl amine),poly(vinyl amine), poly(amidoamine), and poly(2-aminoethylmethacrylate).
 62. The multilayered interpolyelectrolyte complex ofclaim 58 wherein the one or more removable functional groups in thefirst and second interpolyelectrolyte complexes, independently from oneanother, are alkyl groups having 1 to 12 carbon atoms.
 63. Themultilayered interpolyelectrolyte complex of claim 62 wherein the one ormore removable functional groups are methyl, ethyl, propyl, butyl,pentyl, hexyl groups or combinations thereof.
 64. The multilayeredinterpolyelectrolyte complex of claim 58 wherein the one or more anionsin the first and second interpolyelectrolyte complexes, independentlyfrom one another, comprise a nucleic acid.
 65. The multilayeredinterpolyelectrolyte complex of claim 64 wherein the nucleic acidcomprises RNA or DNA.
 66. The multilayered interpolyelectrolyte complexof claim 64 wherein the nucleic acid encodes a protein or a functionalfragment thereof.
 67. The multilayered interpolyelectrolyte complex ofclaim 64 wherein the nucleic acid is a plasmid.
 68. The multilayeredinterpolyelectrolyte complex of claim 58 wherein the one or more anionsin the first and second interpolyelectrolyte complexes, independentlyfrom one another, comprise a therapeutic molecule, a diagnosticmolecule, a peptide, or a carbohydrate.
 69. The multilayeredinterpolyelectrolyte complex of claim 58 wherein the one or more anionsin the first and second interpolyelectrolyte complexes, independentlyfrom one another, comprise a small molecule.
 70. The multilayeredinterpolyelectrolyte complex of claim 58 further comprising one or moreadditional layers, each layer comprising an additionalinterpolyelectrolyte complex comprising the dynamic charge statecationic polymer and said one or more anions.
 71. A multilayeredinterpolyelectrolyte complex comprising a first layer comprising a firstinterpolyelectrolyte complex and a second layer comprising a secondinterpolyelectrolyte complex, wherein each of the first and secondinterpolyelectrolyte complexes, independently from one another,comprise: a. a dynamic charge state cationic polymer having a polymericbackbone formed from monomeric units, and five to 100,000 removablefunctional groups attached to the polymeric backbone through five to100,000 labile linkages, wherein the five to 100,000 removablefunctional groups are selected from the group consisting of an alkyl,alkenyl, alkynyl, cycloalkyl, heterocyclic, aryl, heteroaryl, amino,alkylamino, cyano, amide, and carbamoyl group; and b. one or more anionscomplexed to said cationic polymer; wherein the overall positive chargeof the cationic polymer decreases when any of the five to 100,000 of theremovable functional groups is removed from the polymeric backbone,thereby causing said one or more anions to disassociate from thecationic polymer, wherein the cationic polymer and one or more anions inthe first interpolyelectrolyte complex can be the same or different fromthe cationic polymer and one or more anions in the secondinterpolyelectrolyte complex.
 72. The multilayered interpolyelectrolytecomplex of claim 71 wherein the cationic polymer and one or more anionsin the first interpolyelectrolyte complex are the same as the cationicpolymer and one or more anions in the second interpolyelectrolytecomplex.
 73. The multilayered interpolyelectrolyte complex of claim 71wherein the cationic polymer and one or more anions in the firstinterpolyelectrolyte complex are different from the cationic polymer andone or more anions in the second interpolyelectrolyte complex.
 74. Themultilayered interpolyelectrolyte complex of claim 71 wherein thepolymeric backbone in the first and second interpolyelectrolytecomplexes, independently from one another, are selected from the groupconsisting of polyethylene imine, polylysine, polyornithine,poly(propylene imine), poly(allyl amine), poly(vinyl amine),poly(amidoamine), and poly(2-aminoethyl methacrylate).
 75. Themultilayered interpolyelectrolyte complex of claim 71 wherein the fiveto 100,000 removable functional groups in the first and secondinterpolyelectrolyte complexes, independently from one another, areselected from the group consisting of methyl, ethyl, propyl, butyl,pentyl, hexyl groups and combinations thereof.
 76. The multilayeredinterpolyelectrolyte complex of claim 71 wherein at least one of thefirst and second interpolyelectrolyte complexes comprise a dynamiccharge state cationic polymer having the formula:

wherein n is an integer ranging from 5 to 100,000, x is an integer, y isan integer, wherein the mole percent of y ranges from 10 percent to 100percent based on the total amount of x and y, and R is a removablefunctional group selected from an alkyl, alkenyl, alkynyl, cycloalkyl,heterocyclic, aryl, or heteroaryl group.
 77. The multilayeredinterpolyelectrolyte complex of claim 76 wherein R is an alkyl grouphaving 1 to 12 carbon atoms.
 78. The multilayered interpolyelectrolytecomplex of claim 76 is a methyl, ethyl, propyl, butyl, pentyl, hexylgroup or combinations thereof.
 79. The multilayered interpolyelectrolytecomplex of claim 71 further comprising one or more additional layers,each layer comprising an additional interpolyelectrolyte complexcomprising the dynamic charge state cationic polymer and said one ormore anions.
 80. A multilayered interpolyelectrolyte complex comprisinga first layer comprising a first interpolyelectrolyte complex and asecond layer comprising a second interpolyelectrolyte complex, whereineach of the first and second interpolyelectrolyte complexes,independently from one another, comprise: a. a dynamic charge statecationic polymer having a polymeric backbone formed from monomericunits, and one or more removable functional groups attached to thepolymeric backbone through one or more labile linkages, wherein the molepercent of monomeric units of the polymeric backbone attached to the oneor more removable functional groups is between about 10 percent to about100 percent, wherein the one or more removable functional groups areselected from the group consisting of an alkyl, alkenyl, alkynyl,cycloalkyl, heterocyclic, aryl, heteroaryl, amino, alkylamino, cyano,amide, and carbamoyl group; and b. one or more anions complexed to saidcationic polymer, wherein the overall positive charge of the cationicpolymer decreases when one or more of the removable functional groups isremoved from the polymeric backbone, thereby causing said one or moreanions to disassociate from the cationic polymer; wherein the cationicpolymer and one or more anions in the first interpolyelectrolyte complexcan be the same or different from the cationic polymer and one or moreanions in the second interpolyelectrolyte complex.
 81. The multilayeredinterpolyelectrolyte complex of claim 80 wherein the mole percent ofmonomeric units of the polymeric backbone attached to the one or moreremovable functional groups is between about 30 percent to 100 percent.82. The multilayered interpolyelectrolyte complex of claim 80 whereinthe mole percent of monomeric units of the polymeric backbone attachedto the one or more removable functional groups is between about 50percent to 100 percent.
 83. The multilayered interpolyelectrolytecomplex of claim 80 wherein the mole percent of monomeric units of thepolymeric backbone attached to the one or more removable functionalgroups is between about 70 percent to 100 percent.
 84. The multilayeredinterpolyelectrolyte complex of claim 80 wherein the one or moreremovable functional groups in the first and second interpolyelectrolytecomplexes, independently from one another, are selected from the groupconsisting of methyl, ethyl, propyl, butyl, pentyl, hexyl groups andcombinations thereof.
 85. The multilayered interpolyelectrolyte complexof claim 80 wherein the polymeric backbone in the first and secondinterpolyelectrolyte complexes, independently from one another, areselected from the group consisting of polyethylene imine, polylysine,polyornithine, poly(propylene imine), poly(allyl amine), poly(vinylamine), poly(amidoamine), and poly(2-aminoethyl methacrylate).
 86. Themultilayered interpolyelectrolyte complex of claim 80 further comprisingone or more additional layers, each layer comprising an additionalinterpolyelectrolyte complex comprising the dynamic charge statecationic polymer and said one or more anions.